CN116529371A

CN116529371A - Method and system for isolating RNA from self-collected small volume samples

Info

Publication number: CN116529371A
Application number: CN202180058308.XA
Authority: CN
Inventors: R·B·达内尔; D·奥林奇
Original assignee: Rockefeller University
Current assignee: Rockefeller University
Priority date: 2020-05-29
Filing date: 2021-05-28
Publication date: 2023-08-01

Abstract

The present invention provides methods for isolating and characterizing nucleic acids, particularly RNA, from small volumes and from collected samples, including finger prick blood samples, swabs, and saliva samples. The derivatized RNA is intact and of sufficient quality and quantity for RNA analysis, longitudinal RNA sequencing, and whole transcriptomic profiling.

Description

Method and system for isolating RNA from self-collected small volume samples

Technical Field

The present invention relates generally to methods for isolating and characterizing nucleic acids, particularly RNA, from small volumes and from collected samples, including finger prick blood samples, swabs, and saliva samples. The derivatized RNA is intact and of sufficient quality and quantity for RNA analysis, longitudinal RNA sequencing, and whole transcriptomic profiling.

Background

RNA analysis, longitudinal RNA sequencing and whole transcriptome spectroscopy are useful tools for identifying and analyzing biomarkers of disease, infection, exposure, susceptibility, drug response and toxicity (Frank MO et al (2019) BMC Medical Genetics 12:56; doi.org/10.1186/s12920-019-0500-0; casamassii A et al (2017) Int J Mol Sci 18 (8): 1652; sheid AD et al (2018) J Immunol 200:1817-1928). In some disease and cancer studies, solid tissue or tumor samples are used, however this is not practical in clinical studies or continuous monitoring. Due to its non-invasive collection and availability, peripheral blood has advantages in biomarker assessment and discovery, especially compared to solid tissue samples. Many studies have shown that transcriptomic changes in peripheral Blood can be used as biomarkers for infection, exposure to xenobiotics, response to treatment or vaccine, or as indicators of pathological changes in other tissues (Bujoining PR et al (2007) Proc Natl Acad Sci (46): 18211-6; ramilo O et al (2007) Blood 109:2066-2077; mejias A et al (2013) PLoSMed 10 (11): e1001549; hecker M et al (2013) molecular Neurobiol 48:737-756; querec TD et al (2009) Nat Immunol 10:116-125).

There are a number of significant technical challenges to using whole blood for transcriptomic profiling and RNA assessment, including RNA degradation and transcriptomic changes that occur rapidly after blood is drawn from a subject. Traditional agents, such as citrate, heparin and EDTA, inhibit blood clotting but fail to stabilize mRNA transcripts, and altered gene regulation has been observed in whole blood samples, especially when RNA is not immediately isolated (Debey S et al (2004) pharmacogenomics 4:193-207; rainen L et al (2002) Clin Chem 48:1883-1890). Furthermore, most of the RNA in whole blood encodes globulin, and sequencing that does not take this into account may produce low complexity results (i.e., mainly globulin mRNA, with little other unique or rare mRNA species).

Blood RNA stabilization methods have been developed to address the problem of RNA analysis of whole blood and peripheral blood samples (Asare AL et AL (2008) BMC Genomics 9:474; rainen L (2002) Clin Chem48:1883-1890; chai V et AL (2005) J Clin Lab Anal 19_182-188). Including PAXgene ^TM Blood RNA System (Qiagen) and Tempus ^TM System (applied biosystems company (Applied Biosystems)). In both systems, blood is lysed immediately after collection into a tube and RNA is stabilized with a specific reagent. PAX blood collection tube systems and stabilization buffers are described in U.S. Pat. Nos. 6,602,718 and 6,617,170. Aspects of the stabilizing reagent based on Tempus guanidine salts are provided in 5,972,613. Both systems are designed for the requirement of 2.5ml or 3ml whole blood, require venipuncture, and are not suitable for small blood samples, e.g. from laboratory animals, infants or any suitable self-collection means.

Finger lancing is a practical and minimally invasive sample collection method that is widely used in routine clinical practice and can be practiced outside of the clinical setting. For example, millions of individuals use finger stick sampling to collect small daily blood volumes to monitor sugar or glucose levels. Finger stick blood collection is also valuable for subjects who are often difficult to collect by venipuncture, such as for infants and young children, elderly or venous impaired individuals, intravenous drug takers and individuals who are too obese, in-situ studies in remote and less developed areas, military subjects or physical activity athletes, or other situations, such as where a rapid sample is required or adapted or where a large sample (including from many people) needs to be collected in a short period of time.

However, the RNA collection and analysis systems currently in use and available are not designed for small volume samples, such as lancing samples or samples of one or more drops of blood. The collection of small volumes of blood by finger prick is particularly suitable for high frequency or repeated sample collection, for example, to be able to monitor the health and disease or infection of an individual. Furthermore, these systems are not suitable for other types of samples, such as nasopharyngeal, nasal or throat swabs or aspirates, which may be important for time and sample volume. These are commonly used for direct and rapid patient assessment of viral infections, particularly respiratory viral infections (e.g., influenza), so that the infection can be rapidly assessed and treated.

In order to achieve and apply RNA isolation, assessment and analysis more widely in a variety of clinical and non-clinical scenarios and situations, there is a need for a method and system for reliably and efficiently sampling and analyzing RNA from small volumes of samples and alternative sample types that can be collected frequently, rapidly, and in large quantities in the field or at home by relatively untrained individuals or non-health professionals or patients. There is a need for a direct and reliable system and method whereby RNA can be isolated from small volumes of sample, self-collected samples, finger stick samples, and evaluated qualitatively and quantitatively with reliable and reliable results, particularly for whole transcriptome analysis and profiling (profiling).

Disclosure of Invention

The present invention relates generally to methods for RNA isolation and RNA profiling and analysis of small volume samples and self-collected samples, wherein the RNA is of sufficient quality and quantity for whole transcriptome analysis and transcriptome profiling. In embodiments of the methods, the small volume sample may be from a patient or individual having or at risk of or suspected of having a disease or infection. In some embodiments, the patient or individual obtains or collects a small volume sample. In some embodiments, the patient or individual is assisted by non-medical personnel at the time of sample collection. In one embodiment, the sample is collected from a patient or individual by a non-medical person (e.g., a spouse, parent, friend, guardian, etc., who has not received medical training or who has not been involved in any medical profession). Importantly, the present invention describes methods for obtaining RNA of sufficient quality and quantity for a variety of analyses, ranging from quantifying individual RNA species to sequencing the entire transcriptome of high complexity.

According to this method, a small volume of sample is collected and combined with an RNA stabilizing solution. In some embodiments, the RNA stabilization solution is capable of lysing cells in a sample and stabilizing RNA contained in the cells or cell lysate of the sample. In some embodiments, the RNA stabilization solution is capable of lysing cells in a sample and stabilizing RNA contained in the cells or cell lysate of the sample in a single step. In embodiments, the sample and the RNA stabilization solution are mixed, vortexed, or shaken upon binding. In some embodiments, the sample may be stored or left at room temperature for several hours or hours prior to refrigeration. In some embodiments, the sample is then stored under refrigerated conditions, such as for a short period of time at about 40°f or about 4 ℃. In some embodiments, the sample is then stored under refrigerated conditions, e.g., for a short period of time at about 40°f or about 4 ℃, for up to one or several days or days. In some embodiments, the sample may be stored or left at room temperature for up to several hours or hours, up to 2 hours, up to 3 or 4 hours, prior to refrigeration. In some embodiments, the sample is then stored under refrigerated conditions, e.g., for a short period of time at about 40°f or about 4 ℃, up to one or more days or days. In some embodiments, the sample is stored in a freezer or at refrigeration temperature (e.g., about 30 or 32F. Or about 0℃.) after collection, after a brief (2-4 hours) storage at room temperature, or after a brief (1-2 days) storage by refrigeration.

The small volume sample may be less than 500. Mu.l, less than 300. Mu.l, less than 250. Mu.l, about 200-300. Mu.l, less than 200. Mu.l, about 100-300. Mu.l, about 150-300. Mu.l, about 100-250. Mu.l, and about 50-300. Mu.l. In one embodiment, the small sample volume is about 100-300. Mu.l.

In some embodiments, the sample is a small volume blood sample, sputum or saliva sample, or a nasal, nasopharyngeal or oropharyngeal swab, wash, or aspirate. In embodiments, the low volume sample is a blood sample and is collected by finger prick or heel prick. In one embodiment, the small volume sample is a blood sample and is collected by finger prick. In embodiments, the finger stick or heel sample may comprise a drop of blood directly from a finger stick or heel stick, and a capillary tube may also be utilized.

In some embodiments, the small volume sample may be less than 500. Mu.l, less than 300. Mu.l, less than 250. Mu.l, about 200-300. Mu.l, less than 200. Mu.l, about 100-300. Mu.l, about 150-300. Mu.l, about 100-250. Mu.l, about 50-300. Mu.l. In some embodiments, the volume is less than 100. Mu.l, less than 50. Mu.l, about 10-20. Mu.l, about 10. Mu.l, as little as 10. Mu.l, or less. In one embodiment, the sample volume is about 100 to 300. Mu.l. In one embodiment, the sample volume is about 50 to 300. Mu.l. In some embodiments, the sample volume is about a drop volume, or one or more drop volumes. In some embodiments, the blood or sample volume is a capillary volume, or is less than a drop volume. The capillary sample volume can be 60-100. Mu.l, 100-200. Mu.l, 5-25. Mu.l, 10-50. Mu.l, less than 10. Mu.l, 1-5. Mu.l.

In some embodiments, the volume of the RNA stabilizing solution is less than 1ml, about 500. Mu.l or less, about 300. Mu.l or less, about 200-300. Mu.l, or about 250. Mu.l, or about 200. Mu.l. In one embodiment, the volume of the RNA stabilizing solution is about 300. Mu.l or less, about 200-300. Mu.l, or about 250. Mu.l, or about 200. Mu.l. In some embodiments, including where the sample volume is very low, e.g., about less than 50. Mu.l, or 10-50. Mu.l, or about 10. Mu.l, or less than 10. Mu.l, the volume of the RNA stabilizing solution is suitably low, e.g., about less than 100. Mu.l, less than 50. Mu.l, less than 25. Mu.l, as little as 10. Mu.l or less.

In some embodiments, the sample is collected into a tube, or wherein the tube or container for receiving a small volume of sample and containing the RNA stabilization solution has a total volume capacity of 1.5ml or less, 1.2ml or less, or 1ml or less, or less than 1ml, or less than 500 μl, or less than 300 μl, or less than 200 μl. In one embodiment, the sample is collected into a tube, or wherein the tube or container for receiving a small volume of sample and containing the RNA stabilization solution has a total volume capacity of 1.5ml or less, such as a microtainer blood collection tube. In one embodiment, the sample is collected in a tube suitable for small volumes, including very small volumes, such as capillaries. In one embodiment, the sample is collected in a capillary tube, which is suitable for small volumes, e.g. less than 100 μl, even for very small volumes, e.g. less than 50 μl, less than 25 μl.

The present invention provides a method for RNA profiling and analysis of a small volume sample from a patient or individual, comprising:

(a) Obtaining one or more small volumes of sample collected by the patient or individual or by non-medical personnel, wherein the sample is collected in or otherwise combined with an RNA stabilizing solution, whereby cells in the sample are lysed and the RNA is stabilized; and

(b) Isolating RNA using a procedure suitable for small volume samples, wherein the amount of any and all solutions or buffers used is reduced and adjusted for small volume samples;

wherein the quality and quantity of RNA is sufficient for whole transcriptome analysis and transcriptome profiling by RNA sequencing (RNAseq).

In an embodiment of the method, RNA is isolated using an operation comprising:

(a) Contacting the sample with a protease to form a protease-treated small volume sample;

(b) Contacting the protease-treated sample with an ethanol or salt solution to form an RNA-containing precipitate, wherein the RNA-containing precipitate is then resuspended in a buffer or solution, or contacting the protease-treated sample with an organic extraction solution to form a solution having an aqueous phase containing RNA and an organic phase;

(c) Contacting the RNA-containing resuspended pellet or the RNA-containing aqueous phase with a dnase to form a dnase-treated resuspended pellet or dnase-treated aqueous phase;

(d) Binding RNA to a silica-based solid phase or column by contacting the resuspended precipitate or aqueous phase with the silica-based solid phase; and

(e) Eluting RNA from the silica-based solid phase, comprising contacting the silica-based solid phase with a solution or buffer to provide isolated RNA;

wherein all buffer and solution volumes are reduced and adjusted for small volumes of sample.

In some embodiments, between steps (b) and (c), the RNA-containing resuspended pellet or the RNA-containing aqueous phase is contacted with a solution or column to remove residual sample cell debris and/or to homogenize the sample cell lysate.

For embodiments using a protease, the protease may be proteinase K. For embodiments using proteinase K, the lysis buffer may also contain a detergent that both inactivates the foreign agent, lyses the cells, and activates proteinase K. Proteinase K is active at 25℃although it can be activated by placing the collected sample in hot tap water (typical tap water is set at up to 120℃F., i.e. about 48 ℃); proteinase K has optimal activity at about 55 ℃).

In an embodiment of the method, RNA is isolated using an operation comprising:

(a) Contacting the sample with an RNA stabilizing solution, wherein the solution has the ability to lyse cells and inactivate extraneous agents;

(b) Contacting the RNA-stabilized solution-treated sample with an ethanol or salt solution to form an RNA-containing precipitate, wherein the RNA-containing precipitate is then resuspended in a buffer or solution, or contacting the RNA-stabilized solution-treated sample with an organic extraction solution to form a solution having an aqueous phase containing RNA and an organic phase;

In an embodiment of the method, RNA is isolated using an operation comprising:

(b) Optionally further contacting the sample with a salt, a reducing agent, and/or a detergent;

(c) Contacting the sample (solution contacted sample) with silica, silica-based solid phase or carboxylated magnetic beads capable of binding RNA and purifying RNA from other components in the sample, and the solution of (a) or (b); and

eluting RNA from the silica or silica-based solid phase or magnetic beads, including contacting the silica, silica-based solid phase or magnetic beads with a solution or buffer to provide isolated RNA;

In one embodiment, the RNA stabilizing solution may be a mixture of chaotropic salts and phenols. In one embodiment, the chaotropic salt can be a guanidine salt or a guanidinium salt. In one embodiment, the RNA stabilizing solution may be a PAXgene RNA stabilizing solution. .

In one embodiment, a detergent may be used to purify out chaotropic salts (e.g., guanidine). Downstream purification as in step (c) may be precipitation, contact with nucleic acid binding solid beads or semi-porous beads (e.g. silica or carboxylated magnetic beads). Modification of the lysis buffer for contact with silica or magnetic beads may be a detergent comprising a salt (e.g. sodium acetate) (e.g. 0.2% myo-aminoacyl), a reducing agent (e.g. dithiothreitol, e.g. 75 mM). Purification the nucleic acid can be purified using a magnet by washing the beads with bound nucleic acid twice in 75-80% ethanol or isopropanol, and then eluting the RNA on the beads in pure RNase-free double distilled water (ddH 2O).

In some embodiments, the sample is a small volume blood sample, sputum or saliva sample, or a nasal, nasopharyngeal or oropharyngeal swab, wash, or aspirate. In some embodiments, the sample is a small volume blood sample. In one embodiment, the small volume sample is a blood sample and is collected by finger prick. In embodiments, the finger stick sample may comprise a drop of blood directly from the finger stick, and a capillary tube may also be utilized.

In some embodiments of the method, the sample volume is less than 500. Mu.l, less than 300. Mu.l, less than 250. Mu.l, about 200-300. Mu.l, less than 200. Mu.l, about 100-300. Mu.l, about 150-300. Mu.l, about 100-250. Mu.l, about 50-300. Mu.l. In one embodiment, the sample volume is about 100 to 300. Mu.l. In some embodiments, the volume is less than 100. Mu.l, less than 50. Mu.l, about 10-20. Mu.l, about 10. Mu.l, as little as 10. Mu.l, or less. In one embodiment, the sample volume is about 50 to 300. Mu.l. In one embodiment, the sample volume is about 50 to 250. Mu.l or about 50 to 200. Mu.l.

In some embodiments of the methods, the buffer and solution volumes are reduced to 20-40% or 20-30% of the volume used to isolate RNA from standard venipuncture blood samples.

In some embodiments, the RNA stabilizing solution is a chaotropic salt (e.g., a solution based on or containing guanidine thiocyanate). In some embodiments, chaotropic salts (e.g., guanidine thiocyanate-based lysis buffers) can also contain detergents that work synergistically to inactivate exotic agents, lyse cells. The detergent may comprise sarcosyl, SDS or other ionic or nonionic detergents. Kits/lysis solutions comprising chaotropic salts (e.g., with or without detergent, guanidine thiocyanate based lysis buffers) are stable, even for years. They can be transported and used at room temperature. They are less toxic than household bleaches and can be mailed according to suitable criteria or such.

In some embodiments, any buffer or solution is made, prepared, or generated with rnase-free water or buffer.

In embodiments of the methods, any suitable and effective protease is used. Suitable proteases are well known and available in the art. In embodiments, the protease is proteinase K. In some embodiments, the sample is contacted and treated with the protease at a temperature above room temperature. In embodiments, the sample and protease are heated for protease treatment. In one embodiment, the sample and protease are heated to 50-60℃or incubated at a temperature of 50-60 ℃. In one embodiment, the sample and protease are heated to or incubated at 55 ℃.

According to an embodiment of the method, the method further comprises sequencing the RNA. The RNA can be sequenced using any suitable or recognized method, procedure, system, or kit, including manual, semi-automated, or automated methods, systems, or kits. In some embodiments, a kit (e.g., illumina TruSeq or Kapa Hyper Prep kit) is used.

In one embodiment, the isolated RNA is converted to cDNA. In some embodiments, the isolated RNA is converted to cDNA and can be cloned or prepared therefrom or comprise or be based on a cDNA library.

In some embodiments, a substantial amount of RNA species or RNA species not of interest are removed prior to sequencing.

In embodiments, the globin mRNA, ribosomal RNA, or species-specific RNA is removed prior to sequencing. Methods, systems and kits for removing globulin and/or ribosomal RNA are well known and available to those of skill in the art. In some embodiments, a system or kit may be used, such as Blobinzero (Yingda), ribo-Zero Gold, truSeq Total RNA library preparation, ribo-Zero Global in, GLOBINclear kit (Semer Fielder), QIAseqFastSelect RNA removal kit (Kanji). In some embodiments, species-specific probes may be used to select certain RNAs.

In embodiments or methods, the patient or individual has or is at risk of having or is suspected of having a disease or infection.

In some embodiments, the method is for longitudinal screening by RNA profiling and analysis of a small volume sample from one or more patients or individuals who have or are at risk of or suspected of having a disease or infection. In embodiments, small volumes of sample are collected continuously or regularly or in specified increments of hours, days, weeks, or months. In embodiments, small volumes of sample are collected continuously or regularly by finger prick or in specified increments of hours, days, weeks or months.

In some embodiments, a small volume sample may be collected or otherwise collected at the onset of symptoms (e.g., one or more symptoms or a recognized indicative parameter associated with a disease or associated with an infection). The disease may be an acute or chronic disease. The disease may be a recurrent and/or remitting disease. The infection may be a bacterial or viral infection. The infection may be caused by known or unknown infectious agents. Infection may be caused by known or unknown viral or bacterial masses.

According to embodiments of the present invention, systems and kits for use and application of the method are provided.

In an embodiment, a system or kit for RNA profiling and analysis of a small volume sample from a patient or individual is provided, comprising:

(a) Means for collecting small volumes of sample by the patient or individual or non-medical personnel at their own discretion, including lancets, swabs or containers for washing, spitting or aspiration;

(b) A tube or container for receiving a small volume of sample to be collected and containing a volume of RNA stabilization solution, whereby cells in the sample are lysed and RNA is stabilized; and

(c) One or more suitable labels are used to indicate the name or identity of the patient or individual, the date of sample collection, and the time of sample collection.

In embodiments, the system or kit further comprises an envelope or mailing container for transporting the sample to a laboratory or facility for RNA isolation and analysis.

In some embodiments, the system or kit may be used to perform longitudinal RNA spectroscopy and analysis of multiple small volume samples continuously collected from a patient or individual over days, weeks, or months, including:

(a) A multi-tool stack for collecting small volumes of samples by a patient or individual or by non-medical personnel, each comprising a lancet, swab, or container for washing, spitting, or aspiration;

(b) A multi-tube or container stack, each separately for receiving a small volume of sample to be collected and containing a volume of RNA stabilizing solution, whereby cells in the sample are lysed and the RNA is stabilized;

(c) A plurality of suitable labels for designating the name or identity of the patient or individual, the date of sample collection, and the time of sample collection; and

(d) Multiple envelopes or mailing containers for transporting each sample or several samples to a laboratory or facility for RNA isolation and analysis.

In some embodiments described in the system or kit, the volume of the RNA stabilizing solution is less than 1ml, about 500. Mu.l or less, about 300. Mu.l or less, about 200-300. Mu.l, or about 250. Mu.l. In one embodiment, the volume of the RNA stabilizing solution is about 300. Mu.l or less, about 200-300. Mu.l, or about 250. Mu.l. In some embodiments, including where the sample volume is very low, e.g., about less than 50. Mu.l, or 10-50. Mu.l, or about 10. Mu.l, or less than 10. Mu.l, the volume of the RNA stabilizing solution is suitably low, e.g., about less than 100. Mu.l, less than 50. Mu.l, less than 25. Mu.l, as little as 10. Mu.l or less.

In some embodiments described in the systems or kits, the tube or container for receiving a small volume of sample and containing the RNA stabilization solution has a total volume capacity of 1.5ml or less, 1.2ml or less, or 1ml or less. In some embodiments, the tube or container for receiving a small volume of sample and containing the RNA stabilization solution is a tube suitable for small volumes, including very small volumes, such as capillaries. In one embodiment, the tube or container is a capillary tube suitable for small volumes, e.g. less than 100 μl, even for very small volumes, e.g. less than 50 μl, less than 25 μl.

Other objects and advantages will become apparent to those skilled in the art upon review of the following detailed description of the invention, which is made with reference to the following illustrative drawings and appended claims.

Brief description of the drawings

The patent or patent application contains at least one color drawing. Copies of this patent or patent application publication with color drawings will be provided by the U.S. patent and trademark office upon request and payment of the necessary fee.

Figure 1 depicts a study overview and validation of family assessment of disease activity and gene expression. A. Clinical data collection and RNA analysis over time. Summary of the study of clinical data and sample collection over time. B. Clinical and patient report assessment of disease activity. The correlation between disease activity scores measured at the clinic (DAS 28) and at home (RAPID 3 questionnaire) comes from index patients. C. Clinical blood count and RNASeq inferred blood count. Neutrophil, lymphocyte and monocyte counts (n=38 paired samples) measured from paired clinical whole blood counts of venipuncture blood collection and blood counts deduced from cibertortx of RNAseq data of finger puncture blood collection.

FIG. 2 depicts the mass and quantity of RNA in terms of fixative volumes. 3 drops of blood collected with a 21-gauge needle (Lancet) were added to microtainer blood collection tubes pre-filled with 250. Mu.l, 500. Mu.l or 750. Mu.l PAX gene fixative. Samples were stored at room temperature for 3 days, then RNA was extracted using the PAX gene RNA kit, and RIN scores and the amount of RNA were assessed using the Agilent 2100 picochip. Padj=ANOVA followed by Dunnett multiple comparison test (Dunnett's multiple comparisons test) with 250 μl as reference group.

FIG. 3 depicts RNA quality and quantity over time at room temperature. 100 μ1 whole blood was added to a microtainer tube pre-loaded with 250 μ1PAX gene fixative and frozen after incubation for 2 hours, 3 days or 7 days at room temperature. RNA was extracted with PAX gene RNA kit and washed and eluted with downscaling, and RNA picochip was used to evaluate RIN score and number of RNA using the Agilent 2100 bioanalyzer. Padj=anova followed by Dunnett multiple comparison test (Dunnett's multiple comparisons test) with day 0 as reference group.

FIG. 4 depicts RNA quality and quantity of fresh and mailed samples. 100 μ1 whole blood was added to a microtainer tube pre-loaded with 250 μ1PAX gene fixative and incubated at room temperature for 2 hours or post-mailed frozen. RNA was extracted using PAX gene RNA kit and RIN score and quantity of RNA was assessed using the Agilent 2100 bioanalyzer RNA picochip.

FIG. 5 depicts the quality and quantity of RNA in terms of extraction and washing volumes. 3 drops of blood collected with a 21-gauge needle were added to a microtainer tube previously filled with 250. Mu.l of PAX gene fixative. Samples were stored at room temperature for 3 days, then RNA was extracted using the PAXgeneRNA kit or PAX protocol in a scaled down form according to the manufacturer's instructions, all washes and elutions were performed using approximately 25% of the recommended volume. RIN score and RNA quantity were assessed using an Agilent 2100 bioanalyzer RNA picochip. P = unpaired double sided t-test.

FIG. 6 depicts the quality and quantity of RNA with and without the TriZol reagent extraction step. The mailed patient finger stick samples were stored in PAXgeneRNA buffer at-80 ℃.142 samples were RNA extracted by PAXgeneRNA extraction and low volume washed, and 13 samples were thawed and mixed with 700. Mu.l Trizol-LS and 250. Mu.l chloroform. After centrifugation, the top layer was precipitated with isopropanol and glycogen, and washed with 80% cold ethanol, centrifuged and the precipitate dried, resuspended in PBS and then purified using Luo Shigao pure separation kit. P-values represent significance of unpaired T-test.

FIG. 7 depicts cycle time comparisons of HbgA2, 18S RNA and TNF alpha after Globinzero depletion. Since ribosomal and hemoglobin RNAs represent about 98% and 70% of RNA in whole blood, respectively, we tested standard commercial kits for removing these RNAs prior to RNAseq. 4ml of heparinized blood were treated and stimulated with 1. Mu.g/ml LPS, or untreated and incubated for 1 hour at 37 ℃. Then, 250. Mu.l of unstimulated or stimulated blood sample was placed in 250. Mu.l PAXgene fixative and placed in duplicate microtainer blood collection tubes. After RNA extraction, samples were either not depleted (left panel) or were depleted with globinlero kit (right panel), and then quantitative PCR was performed to detect the expression of hemoglobin A2, 18S RNA or tnfα mRNA. The globinlzero kit depletes hemoglobin A2 and 18S ribosomal RNAs (average cycle time increases from 11 to 28 and 10 to 30, respectively) while retaining tnfα mRNA. The P-value represents the result of a common one-way analysis of variance and Tukey multiple comparison test.

FIG. 8 provides RNASeq QC indices for RNAs with different mass fractions prepared with the Illumina TruSeq or Kapa Hyper Prep kit. A. (left panel): mapping, unique mapping, and distribution of duplicate readings. B. (right panel): the tag distribution of UTR (untranslated region), intergenic region, introns and CDS (coding sequence) assigned to whole blood RNA samples prepared with Illumina TruSeq or Kapa Hyper Prep kit, with various input RNA quality and quantity. Illumina TruSeq library preparation showed increased mapping of coding sequences and fewer intergenic region reads and was ultimately used in downstream RNA sequencing experiments.

Figure 9 provides clinical and transcriptional signatures indexing RA episodes in patients. A. Patient disease activity is indexed over time. Disease activity (RAPID 3 questionnaire, n=356) was indexed for four years in patients. Time points are colored according to disease activity categories. B. Differential expression of genes at onset. Volcanic plots of onset (n=46) versus baseline (n=33) differential gene expression, with statistical significance (-log 10 (FDR)) for fold change (log 2 (FC)) plotted (grey dots are non-significant genes, i.e., FDR >0.1, red for FDR <0.1, log2 fold change >0, blue for FDR <0.01, log2 fold change < 0). The pathway of enrichment at the time of onset is significantly increased (c.) (pathway at the time of onset is increased) or the gene is decreased (d.) (pathway at the time of onset is decreased) relative to baseline.

Figure 10 provides transcriptional signatures of pre-symptomatic immune activation at RA onset. A. Disease activity score (in days) to onset over time. Boxes represent disease activity from day-56 to day +28 over time to onset. The vertical arrow (in a-D) indicates the onset of the episode. 2791 hierarchical clustering of z scores of significantly differentially expressed genes over time to onset. Clusters with statistical significance are labeled with color. AC2 and AC3 refer to clusters that change pre-to episodes. C. Specific illustrations of cluster 1, pre-cluster 2 (AC 2) and pre-cluster 3 (AC 3) genes over time to seizure in fig. 3B. D. Average normalized clustered gene expression over time to onset. The light grey line indicates the expression of individual genes in the cluster. The horizontal dashed line represents average baseline gene expression (weeks-8 to-4). The vertical dashed line represents the onset of the episode. E. Pathways enriched in clusters 1, AC2 and AC 3.

FIG. 11 PRIME cells expressed the AC3 gene. A. Synovial cell subtype marker gene clusters identified in blood (fig. 3A). Enrichment fraction of 200 single cell RNAseq marker genes from 18 synovial subcellular types. The dashed line represents the significance threshold (FDR <0.05 or-log 10 FDR > 1.3). B. Over time to onset, the average normalized gene expression of common genes for synovial lower fibroblasts (cd34+, dkk+ and HLA-dra+ fibroblasts) and AC3 in blood and 95% confidence intervals (dashed lines vertically indicate onset of onset). Error bars represent confidence intervals. Venn diagram of AC3 gene reduced during seizure in 4 patients. D. Flow cytometry of blood samples from 19 RA patients and 18 Healthy Volunteers (HV). The percentage of TOPRO- (live)/CD 31-cells PDPN+/CD 45-cells is shown. The P value represents the result of the double sided t-test. Log2 fold change of prime cells (flow sorted CD45-/CD31-/pdpn+ cells) versus hematopoietic cells (flow sorted cd45+) expressed AC3 genes and Log2 fold change of input cells (stained PBMC but not flow sorted) versus hematopoietic cells (flow sorted cd45+) were used as technical controls of flow sort pressure.

Fig. 12 depicts reproducible changes in differentially expressed seizure genes in repeated seizures. A. Patient disease activity (RAPID 3) is indexed over time. The dots of the upper graph distribute the coloring according to the disease activity. The dots of the lower panel are colored according to clinical seizure event numbers. B. Unsupervised hierarchical clustering of gene differential expression between baseline and onset. The upper bar indicates the dispensing of colored samples according to disease activity. The lower bar represents samples colored according to clinical seizure event numbers. The data shows differentially expressed seizure genes are represented by multiple clinical events.

FIG. 13 depicts the technical control of the Log2 fold change of various synovial single cell RNAseq marker genes relative to hematopoietic cells (flow-sorted CD45+) and Log2 fold change of input cells (stained PBMC but not flow-sorted) relative to hematopoietic cells (flow-sorted CD45+) in a sorted PRIME cell expressing the synovial fibroblast gene PRIME cell (flow-sorted CD45-/CD31-/PDPN+ cells) as flow sort pressure. These data show that single cell marker genes of fibroblasts (SC-F1, SC-F2, SC-F3, SC-F4) but not B cells (SC-B1-4), macrophages (SC-M1-4) or T cells (SC-T1-6) are enriched in sorted PRIME cells. Fibroblast genes (e.g., markers) are the only set of synovial cell marker genes that are enriched in PRIME cells.

FIG. 14 depicts sorted PRIME cells expressing classical synovial fibroblast genes. Volcanic plot of Log10 (-padj) versus Log2 fold change of PRIME cells (flow sorted CD45-/CD31-/pdpn+ cells) versus hematopoietic cells (flow sorted cd45+). Classical fibroblast genes are significantly increased in PRIME cells relative to hematopoietic cells.

Detailed Description

Conventional molecular biology, microbiology and recombinant DNA techniques within the skill of the art can be used in accordance with the present invention. These techniques are well described in the literature. See, for example, sambrook et al, molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual) (1989); new Programming guidelines for molecular biology (Current Protocols in Molecular Biology) volume I-III [ Ausubel, R.M. (1994) ]; cell biology: laboratory Manual (Cell Biology: A Laboratory Handbook) volume I-III [ J.E.Celis, eds. (1994) ]; new Instructions for immunology, volume I-III (Current Protocols in Immunology) (Coligan, J.E. Ind. (1994)); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984); nucleic acid hybridization (Nucleic Acid Hybridization) [ B.D.Hames and S.J.Higgins, eds. (1985) ]; transcription and translation (Transcription And Translation) [ b.d.hames and s.j.higgins, eds. (1984) ]; animal cell culture (Animal Cell Culture) [ R.I. Freshney code (1986) ]; immobilized cells and enzymes (Immobilized Cells And Enzymes) [ IRL Press (1986) ]; erbal, guidelines for molecular cloning practice (A Practical Guide To Molecular Cloning) (1984).

Thus, if present herein, the following terms shall have the following definitions.

The term "rheumatoid arthritis (rheumatoid arthritis)" or "RA" refers to a chronic disease that is immune-mediated and inflammatory, is an autoimmune disease, affects the endo-articular membranes, results in pain, stiffness, swelling, and reduced joint movement in the joints, and can ultimately lead to bone erosion and joint deformity (joint deformity). RA is a systemic autoimmune disease characterized by simultaneous inflammation of the synovium of multiple joints.

"RA episode" or "episode" refers to a surge in immune-mediated and/or inflammatory activity that is periodically experienced by a patient with RA. During the onset, fatigue and joint symptoms (e.g., pain, swelling, and stiffness) temporarily increase. Attacks are periods of increased disease activity during which the arthritic symptoms of a person, typically including joint pain, swelling and stiffness, are more severe. RA episodes may involve exacerbation of any disease symptoms, but most commonly include strong stiffness of the joints. People with RA report common symptoms of these episodes: joint stiffness increases, general pain increases, difficulty in completing daily tasks increases, swelling (e.g., resulting in a shoe without a foot), intense fatigue, flu-like symptoms.

As used herein, "RNA" is defined as at least two ribonucleotides covalently linked together. The RNA may be any type of RNA. Examples include mRNA, tRNA, rRNA, shRNA, circRNA, scaRNA, scRNA, snRNA, siRNA or Piwi-interacting RNA (Piwi-interacting RNA), or primary miRNA, precursor miRNA, miRNA, snoRNA, long ncRNA, anti-miRNA, precursor and any variants thereof. Further examples of RNAs include viral RNAs, or RNA sequences derived from viral genomes. Still further examples include bacterial RNAs. The RNA may be single-stranded or double-stranded, or may contain both double-stranded and single-stranded sequence portions. RNA can be synthesized as a single stranded molecule or expressed in cells (in vitro or in vivo) using synthetic genes. RNA may be obtained by chemical synthesis methods or by recombinant methods.

RNA may also encompass the described single-stranded complementary strand. Many variants of RNA can be used for the same purpose as a given RNA. Thus, RNA also encompasses substantially identical RNA and complements thereof. The single strand provides a probe that hybridizes to the target sequence under stringent hybridization conditions. Thus, RNA also encompasses probes that hybridize under stringent hybridization conditions.

As used herein, "pg" means picogram, "ng" means nanogram, "ug" or "μg" means microgram, "mg" means milligram, "ul" or "μl" means microliter, "ml" means milliliter, and "l" means liter.

A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that serves as an autonomous unit of DNA replication in vivo (i.e., capable of replication under its own control).

The vector is a replicon, such as a plasmid, phage, or cosmid, in which another DNA segment is ligated to allow replication of the ligated segment.

"DNA molecule" refers to a polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its single-stranded form or in a double-stranded helix. The term refers only to the primary and secondary structure of the molecule and is not limited to any particular tertiary form. Thus, the term includes, inter alia, double stranded DNA found in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, chromosomes, and the like. In discussing the structure of a particular double-stranded DNA molecule, the sequence may be given only the sequence in the 5 'to 3' direction along the non-transcribed strand of the DNA (i.e., the strand having a sequence homologous to the mRNA), as is normal practice, and is described herein. "origin of replication" refers to a DNA sequence involved in DNA synthesis.

A DNA "coding sequence" is a double-stranded DNA sequence that is transcribed and translated into a polypeptide in vivo under the control of appropriate regulatory sequences. The boundaries of the coding sequence are defined by a start codon at the 5 '(amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. Coding sequences may include, but are not limited to: prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. Polyadenylation signals and transcription termination sequences are typically located 3' to the coding sequence.

Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.

The term "oligonucleotide" as used herein refers to a probe of the invention, defined as a molecule consisting of two or more ribonucleotides, preferably more than three. The exact size depends on many factors, which in turn depend on the final function and use of the oligonucleotide.

The term "primer" as used herein refers to a synthetically produced oligonucleotide that is capable of acting as a point of initiation of synthesis when placed under conditions that induce synthesis of primer extension products complementary to a nucleic acid strand, i.e., in the presence of nucleotides and an inducer (e.g., a DNA polymerase) and at a suitable temperature and pH. The primer may be single stranded and must be long enough to prime the synthesis of the desired extension product in the presence of the inducer. The exact length of the primer depends on many factors, including temperature, primer source and use of the method. For example, for diagnostic applications, an oligonucleotide primer will typically comprise 15-25 or more nucleotides, although it may comprise fewer nucleotides, depending on the complexity of the target sequence.

The primers herein are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize to their respective strands. Thus, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences may be inserted into the primer, provided that the primer sequence has sufficient complementarity to the sequence of the strand to hybridize thereto and thereby form a template for synthesis of the extension product.

A "protease" as defined herein is an enzyme capable of hydrolysing peptide bonds. Conventional proteases may be used. Proteinase K is an example. It is preferred that the specific activity of the protease is high to degrade proteins in the possibly protein-rich sample and to protect the RNA from ribonucleases. The specific activity of the protease in the mixture of biological sample and denaturing solution, as determined by the Chromozym assay, is, for example, at least about 0.1U/ml, at least about 1U/ml, at least about 2.5U/ml, at least about 5U/ml, or at least about 10U/ml. In another embodiment, the specific activity of the protease in the mixture is between 0.1 and 1000U/ml.

Reference in the specification to "one embodiment," "an embodiment," "one example," or "an example" means that a particular feature, structure, or characteristic described in connection with the embodiment or example is included in at least one implementation of the embodiment. Thus, the appearances of the phrases "in one embodiment," "in an embodiment," "an example," or "an example" in various places throughout this specification are not necessarily all referring to the same embodiment or example. Furthermore, the particular features, structures, or characteristics may be combined in any suitable combination and/or sub-combination in one or more implementations or embodiments. Furthermore, it should be understood that the drawings provided herein are for illustrative purposes to one of ordinary skill in the art and that the drawings are not necessarily drawn to scale.

As used herein, the terms "comprising," "including," "having," "containing," and variations thereof mean non-exclusive open-ended. For example, a method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such method, article, or apparatus.

Also, unless expressly stated otherwise, "or" means "or" of compatibility rather than exclusive "or". For example, the case where either of the following satisfies a or B: a true (or present) and B false (or absent), a false (or absent) and B true (or present), and both a and B are true (or present).

In accordance with the present invention, a method, kit and system, and clinical and technical protocols have been established for isolating RNA by repeated home blood sampling of a patient with disease using self-administered small volumes of blood samples that are finger-lanced. The system enables and allows for longitudinal RNA sequencing (RNAseq). In an exemplary set of studies, rheumatoid Arthritis (RA) patients were evaluated, their RNAs were evaluated at a series of time points, and correlated with clinical and physical parameters regarding RA episodes. Samples were obtained from a number (more than 300) time points of eight episodes in four years for one index patient, and more than 200 time points of episodes for three other patients. A sampling method and RNA stabilization and isolation protocol was developed that provides high quality intact RNA. The evaluation results show that RNAseq data from small volume blood finger stick samples correlates with blood cell count of venipuncture blood draw. Transcripts were identified as differentially expressed prior to RA onset. Transcriptomic studies performed on patients prior to RA onset revealed a unique cell type in RA blood, PRIME cells, expected to be activated by B cells several weeks prior to RA onset, and then migrate from the blood into the synovium.

The methods and systems provide and enable longitudinal genomic analysis by whole transcriptome analysis and total RNA sequencing (RNAseq). The studies presented herein demonstrate that the collection system and RNA stabilization and isolation methods allow RNA sampling to obtain valuable and consistent results. In various applications of the system and method, RNA profiling and longitudinal RNAseq analysis using the system and method may reveal dynamic changes leading to the onset of chronic inflammatory disease, provide clinical parameters and susceptibility indicators of disease or infection, reveal mechanisms through RNA activation and/or changes in disease or infection progression or susceptibility thereto, etc.

The present invention relates generally to methods for RNA isolation and RNA profiling and analysis of small volume samples, wherein the RNA is of sufficient quality and quantity for whole transcriptome analysis and transcriptome profiling.

Transcriptomics is a study of the' transcriptome, the initial term referring to the collection of entire transcripts, and is now widely understood to refer to the complete collection of all ribonucleic acid (RNA) molecules expressed in some given entity (e.g., cell, tissue, or organism). Transcriptomics may encompass everything related to RNAs, including their transcription and expression levels, function, location, transport, and degradation. It may also include the structure of the transcript and its parent gene at the start site, 5 'and 3' terminal sequences, splicing patterns and post-transcriptional modifications, and encompasses all types of transcripts, including messenger RNAs (mrnas), micrornas (mirnas) and different types of long non-coding RNAs (lncrnas).

Modern transcriptomics often uses high-throughput methods to analyze the expression of multiple transcripts under different physiological or pathological conditions, and this is rapidly expanding our understanding of the relationship between transcriptomes and a broad range of in-vivo phenotypes. Whole transcriptome analysis and total RNA sequencing (RNA-Seq) detects coding and multiple forms of non-coding RNA, and the goal of total RNA sequencing is to accurately measure gene and transcript abundance and identify known and new features of the transcriptome.

It is important to recognize that different levels of RNA assessment and analysis require different amounts of RNA in terms of yield or quantity and quality. For example, gene expression analysis experiments looking for a rapid snapshot of a highly expressed gene may require only a relatively small or lower quality of RNA, particularly the amount of RNA from the highly expressed gene in the sample is relatively more important (compared to low-expression or relatively rare or small RNA). Evaluation or assessment of target gene expression whether one or more target RNAs are present may require only a relatively small or low quality RNA, particularly in assays where as many specific RNA probes or primer-based isolation procedures as possible may be used. Experiments looking for a more comprehensive view of gene expression and some information about alternative splicing, usually require RNA of moderate quality and quantity. This encompasses most or many published RNA-Seq experiments for mRNA/whole transcriptome sequencing.

In sharp contrast, research or experimentation to find or require deep complete knowledge of the transcriptome to evaluate, identify or assemble new transcripts, accurately measure gene and transcript abundance, or identify known and new characteristics of the transcriptome, requires that the highest quality and quantity of RNA be obtained from the sample. Thus, the methods in which RNA is isolated, even from small or smaller volumes of sample, are not at the highest level in quantity and quality, and are not suitable for use in accurate and complete transcriptome analysis or longitudinal RNA profiling, although they are suitable for use in some RNA assays and studies. The amount of all RNA isolated is insufficient to provide accurate and complete RNA information.

Total RNA-Seq analysis of coding and multiple forms of non-coding RNA to fully understand the transcriptome, and accurate and complete results require high quality RNA in amounts and yields sufficient to provide accurate, full-length and comprehensive RNA sequences representative of the complete transcriptome.

The methods provided herein and the RNAs provided according to the invention are of sufficient quality and quantity that full transcriptome analysis and transcriptome profiling can be performed by RNA sequencing. Known and available RNA isolation methods, if applied in a manner designed for larger volume samples (such as standard venipuncture blood samples, or blood samples of e.g. 2-3 ml), do not yield RNA of suitable quality and quantity for whole transcriptome analysis and transcriptome analysis by RNA sequencing when applied to small volume samples (in particular small blood samples, e.g. from finger pricks, or samples in the blood volume range of 100-300. Mu.l).

In some embodiments, the sample may be a small volume blood sample, sputum or saliva sample, or a nasal, nasopharyngeal or oropharyngeal swab, wash or aspirate. In embodiments, the low volume sample is a blood sample and is collected by finger prick or heel prick. In one embodiment, the small volume sample is a blood sample and is collected by finger prick. In embodiments, the finger stick or heel sample may comprise a drop of blood directly from a finger stick or heel stick, and a capillary tube may also be utilized.

In some embodiments, the small volume sample may be less than 500. Mu.l, less than 300. Mu.l, less than 250. Mu.l, about 200-300. Mu.l, less than 200. Mu.l, about 100-300. Mu.l, about 150-300. Mu.l, about 100-250. Mu.l, about 50-300. Mu.l, about 50-200. Mu.l, about 50-150. Mu.l. In one embodiment, the sample volume is about 100 to 300. Mu.l. In some embodiments, the volume is less than 100. Mu.l, less than 50. Mu.l, about 10-20. Mu.l, about 10. Mu.l, as little as 10. Mu.l, or less. In one embodiment, the sample volume is about 50 to 300. Mu.l. In some embodiments, the sample volume is about a drop volume, or one or more drop volumes. In some embodiments, the blood or sample volume is a capillary volume, or is less than a drop volume. The capillary sample volume can be 60-100. Mu.l, 100-200. Mu.l, 5-25. Mu.l, 10-50. Mu.l, less than 10. Mu.l, 1-5. Mu.l. Capillaries of these volume orders are readily available on the market, for example from Sigma-Aldrich. The selected or preferred volume may be selected among or as preferred.

The small volume sample may be from a patient or individual having or at risk of or suspected of having a disease or infection. In some embodiments, the patient or individual obtains or collects a small volume sample. In some embodiments, the patient or individual is assisted by non-medical personnel at the time of sample collection. In one embodiment, the sample is collected from a patient or individual by a non-medical person (e.g., a spouse, parent, friend, guardian, etc., who has not received medical training or who has not been involved in any medical profession).

According to this method, a small volume of sample is collected and combined with an RNA stabilizing solution. In some embodiments, the RNA stabilization solution is capable of lysing cells in a sample and stabilizing RNA contained in the cells or cell lysate of the sample. In some embodiments, the RNA stabilization solution is capable of lysing cells in a sample and stabilizing RNA contained in the cells or cell lysate of the sample in a single step. In embodiments, the sample and the RNA stabilization solution are mixed, vortexed, or shaken when combined. In some embodiments, the sample may be stored or left at room temperature for several hours or hours prior to refrigeration. In some embodiments, the sample is then stored under refrigerated conditions, such as for a short period of time at about 40°f or about 4 ℃. In some embodiments, the sample is then stored under refrigerated conditions, e.g., for a short period of time at about 40°f or about 4 ℃, up to one or more days or days. In some embodiments, the sample may be stored or left at room temperature for up to several hours or hours, up to 2 hours, up to 3 or 4 hours, prior to refrigeration. In some embodiments, the sample is then stored under refrigerated conditions, e.g., for a short period of time at about 40°f or about 4 ℃, up to one or more days or days. In some embodiments, the sample is stored in a freezer or at refrigeration temperature (e.g., about 30 or 32F. Or about 0℃.) after collection, after a brief (2-4 hours) storage at room temperature, or after a brief (1-2 days) storage by refrigeration.

In some embodiments, the volume of the RNA stabilizing solution is less than 1ml, about 500. Mu.l or less, about 300. Mu.l or less, about 200-300. Mu.l, or about 250. Mu.l. In one embodiment, the volume of the RNA stabilizing solution is about 300. Mu.l or less, about 200-300. Mu.l, or about 250. Mu.l. In some embodiments, including where the sample volume is very low, e.g., about less than 50. Mu.l, or 10-50. Mu.l, or about 10. Mu.l, or less than 10. Mu.l, the volume of the RNA stabilizing solution is suitably low, e.g., about less than 100. Mu.l, less than 50. Mu.l, less than 25. Mu.l, as little as 10. Mu.l or less.

The RNA stabilizing solution may be guanidine salt based. The RNA stabilizing solution may be a PAXgene based solution, a Tempus RNA based solution, a Trizol solution, a QIAzol based solution, or a Dxterity based solution system. Suitable guanidine salt based solutions, such as guanidine thiocyanate solutions, are known. Guanidine salt based solutions and methods have been previously described (e.g., chomczynski P and Sacchi N. (1987) Anal. Biochem. 162:156-159). Some solutions are or may be preferred and more advantageous or appropriate in the method in order to produce RNA of sufficient quality and quantity for RNAseq and transcriptome analysis or longitudinal analysis provided herein.

The sample may be collected into a tube, or wherein the tube or container for receiving a small volume of sample and containing the RNA stabilization solution has a total volume capacity of 1.5ml or less, 1.2ml or less, or 1ml or less, or 500 μl or less. In one embodiment, the sample is collected into a tube, or wherein the tube or container for receiving a small volume of sample and containing the RNA stabilization solution has a total volume capacity of 1.5ml or less, such as a microtainer blood collection tube. In some embodiments, the tube or container for receiving a small volume of sample and containing the RNA stabilization solution is a tube suitable for small volumes, including very small volumes, such as capillaries. In one embodiment, the tube or container is a capillary tube suitable for small volumes, e.g. less than 100 μl, even for very small volumes, e.g. less than 50 μl, less than 25 μl. Tubes or containers of suitable dimensions are well known and available in the art.

RNA can be isolated using a process comprising:

In an embodiment of the method, RNA is isolated using an operation comprising:

(e) Contacting the sample with an RNA stabilizing solution, wherein the solution has the ability to lyse cells and inactivate extraneous agents;

(f) Optionally further contacting the sample with a salt, reducing agent and/or cleaning agent;

(g) Contacting the sample with silica, silica-based solid phase or carboxylated magnetic beads capable of binding RNA and purifying RNA from other components in the sample, and the solution of (a) or (b); and

(h) Eluting RNA from the silica or silica-based solid phase or magnetic beads, including contacting the silica, silica-based solid phase or magnetic beads with a solution or buffer to provide isolated RNA;

In embodiments, all buffer and solution volumes are reduced to about 20-30%, 20-28%, about 25% of the standard venipuncture blood volume, which is about 2.5ml of sample volume. Thus, in commercial kits and methods, while the sample volume is about 1/10 or 10% of the standard blood volume, the buffer and solution is reduced to about 20-30% or about 25%.

In commercial RNA isolation kits (e.g., PAXgene Blood RNA kit), the blood collection tube contains an RNA stabilizing solution that is suitable for a sample volume of about 2.5 ml. The PAXgene blood RNA tube contained 6.9ml of RNA stabilizing solution, which was suitable for about 2.5ml of blood. For PAXgene Blood RNA tubes, the relative ratio of sample volume to RNA stabilizing buffer is about 0.36, or the stabilizing solution volume is about 2.5-3 times the sample volume or about 2.76 times. In the present method, about 500. Mu.l or less, about 300. Mu.l or less, about 200-300. Mu.l, or about 250. Mu.l of RNA stabilizing solution is present or provided for collection of a small volume sample. In the present method, about 500. Mu.l or less, about 300. Mu.l or less, about 200-300. Mu.l or about 250. Mu.l of RNA stabilizing solution is present or provided for collection of small volumes of sample, wherein the sample volume is less than 500. Mu.l, less than 300. Mu.l, less than 250. Mu.l, about 200-300. Mu.l, about 250. Mu.l, less than 200. Mu.l, about 100-300. Mu.l, about 150-300. Mu.l, about 100-250. Mu.l, about 50-300. Mu.l, about 50-200. Mu.l, about 50-150. Mu.l. The sample volume to RNA stabilization buffer ranges from about 5 to about 2 times, from about 5 to about 1 times, from about 3 to about 2 times the sample volume. Although the PAXgene kit contains 6.9ml of RNA stabilizing solution in the blood collection tube, in this method, the sample is bound to about 250. Mu.l or 0.25ml (3-4% relative volume).

In commercial RNA isolation kits (e.g., PAXgene Blood RNA kit), the buffer volume for protease treatment is about 340. Mu.l, containing 300. Mu.l buffer and 40. Mu.l protease. In the present method, the buffer volume for protease treatment is about 74-75. Mu.l, comprising 65. Mu.l of buffer and about 9. Mu.l of protease. The relative volume percent of protease buffer and protease in the present method is about 20-22% or about 22%.

The sample may be a small volume blood sample, a sputum or saliva sample, or a nasal, nasopharyngeal or oropharyngeal swab, wash or aspirate. In some embodiments, the sample is a small volume blood sample. In one embodiment, the small volume sample is a blood sample and is collected by finger prick. In embodiments, the finger stick sample may comprise a drop of blood directly from the finger stick, and a capillary tube may also be utilized.

In some embodiments of the method, the sample volume is less than 500. Mu.l, less than 300. Mu.l, less than 250. Mu.l, about 200-300. Mu.l, less than 200. Mu.l, about 100-300. Mu.l, about 150-300. Mu.l, about 100-250. Mu.l, about 50-300. Mu.l. In one embodiment, the sample volume is about 100-300. Mu.l. In some embodiments, the sample volume is less than 100 μl, less than 50 μl, less than 25 μl, 10 μl, or less.

In some embodiments of the methods, the buffer and solution volumes are reduced to 20-40% or 20-30% or about 25% of the volume used to isolate RNA from a standard venipuncture blood sample (e.g., a 2.5ml or about 2.5ml sample).

In some embodiments, the RNA stabilizing solution is a guanidine thiocyanate-based or containing solution.

In some embodiments, any buffer or solution is made or generated with rnase-free water or buffer.

The purification/separation method may be adapted to make use of fully manual purification. In embodiments of manual purification centrifugation or vacuum manifolds or combinations thereof, for example, to pass the solution through a column, may be used. The purification/separation method may be adapted for or may utilize semi-automatic purification. In semi-automatic purification embodiments, the cleavage step and precipitation or organic extraction step are performed manually, while column purification is performed in an automated manner, for example using an automated liquid handling system. It is contemplated that the isolation method is applied to fully automated purification and is one embodiment of the present invention, wherein all steps are performed using a fully automated system, such as a fully equipped liquid handling system or a fully automated extraction system. It is contemplated that the separation method is applied to fully automated purification and one embodiment thereof, wherein all steps are performed using a fully automated system, such as a fully equipped liquid handling system or a fully automated extraction system. Such fully automated systems are known and available in the art. In some embodiments, the fully automated system is modified to adjust the volumes, reagents, materials used for small volume sample processing.

In embodiments of the method, a commercial kit or RNA purification system is modified. In embodiments, PAXgene blood RNA kits and procedures are modified to be suitable and capable of providing RNA isolation and RNA profiling and analysis of small volume samples, wherein the RNA is of sufficient quality and quantity for whole transcriptome analysis and transcriptome profiling. In embodiments, the Tempus blood RNA system and process is modified to be suitable and capable of providing RNA isolation and RNA profiling and analysis of small volume samples, wherein the RNA is of sufficient quality and quantity for whole transcriptome analysis and transcriptome profiling.

The protocol for manual purification of total RNA from human whole blood collected into PAXgene blood RNA tubes is as follows (handbook 2015):

operation of

1. PAXgene blood RNA tubes were centrifuged at 3000-5000x g for 10 minutes using a horizontal rotor (swing-out rotor).

2. The supernatant was removed by decantation or pipetting. To the pellet was added 4ml of rnase free water and the tube was then closed using a new secondary BD Hemogard lid (provided with the kit).

3. Vortex until the pellet is significantly dissolved, then centrifuge for 10 minutes at 3000-5000x g using a horizontal rotor. The whole supernatant was removed and discarded. Small fragments remaining in the supernatant after vortexing but before centrifugation do not affect the operation.

4. 350 μl of buffer BR1 was added and vortexed until the pellet was significantly dissolved.

5. The sample was pipetted into a 1.5ml microcentrifuge tube. Mu.l of buffer BR2 and 40. Mu.l of proteinase K were added. Vortex mixing for 5 seconds and incubate at 55℃for 10 minutes using a shaking incubator at 400-1400 rpm. After incubation, the temperature of the shake flask was set to 65 ℃ (for step 20).

6. The lysates were aspirated directly into PAXgene crushing centrifugation columns (Syringa) placed in 2ml treatment tubes and centrifuged at maximum speed for 3 minutes (but not more than 20,000Xg).

7. The whole supernatant of the flow-through fraction was carefully transferred to a new 1.5ml microcentrifuge tube without disturbing the pellet in the treatment tube.

8. 350 μl ethanol (96-100% purity grade p.a.) was added. Vortex mixing and briefly centrifuge (500-1000 x g,1-2 seconds) to remove droplets inside the tube cap.

9. Mu.l of the sample was pipetted into a PAXgene RNA centrifuge column (red) placed in a 2ml processing tube and centrifuged at 8000-20,000Xg for 1 min. The column was placed in a new 2ml processing tube and the old processing tube containing the effluent was discarded.

10. The remaining sample was aspirated into a PAXgene RNA centrifuge column and centrifuged at 8000-20,000Xg for 1 min. The column was placed in a new 2ml processing tube and the old processing tube containing the effluent was discarded.

11. Mu.l of buffer BR3 was pipetted into a PAXgene RNA spin column. The column was placed in a new 2ml processing tube by centrifugation at 8000-20,000Xg for 1 min and the old processing tube containing the effluent was discarded.

12. Mu.l of DNase I stock was added to 70. Mu.l of buffer RDD in a 1.5ml microcentrifuge tube. The tube was gently shaken to mix and briefly centrifuged to collect the residual liquid on the sides of the tube.

13. The DNase I incubation mixture (80. Mu.l) was directly pipetted onto the PAXgene RNA spin column membrane and placed on a bench (20-30 ℃) for 15 minutes.

14. 350 μl of buffer BR3 was pipetted into the PAXgene RNA spin column and centrifuged at 8000-20,000Xg for 1 min. The column was placed in a new 2ml processing tube and the old processing tube containing the effluent was discarded.

15. Mu.l of buffer BR4 was pipetted into the PAXgene RNA spin column and centrifuged at 8000-20,000Xg for 1 min. The column was placed in a new 2ml processing tube and the old processing tube containing the effluent was discarded.

16. An additional 500. Mu.l of buffer BR4 was added to the PAXgene RNA spin column. Centrifuging at 8000-20,000Xg for 3 min

17. The treatment tube containing the effluent was discarded and the PAXgene RNA spin column was placed into a new 2ml treatment tube. Centrifuge at 8000-20,000Xg for 1 min.

18. The process tube containing the effluent was discarded. The PAXgene RNA spin column was placed in a 1.5ml microcentrifuge tube and 40. Mu.l of buffer BR5 was directly pipetted onto the PAXgene RNA spin column membrane. Centrifugation was performed at 8000-20,000Xg for 1 min to elute RNA.

19. The elution step was repeated as described using 40 μl buffer BR5 and the same microcentrifuge tube (step 18).

20. The eluate was incubated in a shaking incubator (from step 5) at 65 ℃ for 5 minutes without shaking. Immediately after incubation, the incubation was cooled on ice.

21. If the RNA sample is not used immediately, it is stored at-20℃or-70 ℃.

The PAXgene blood RNA system and method is specific and specifically designed and adapted for a blood sample volume of about 2.5ml, 10 times greater than the blood sample volume treated by the method herein. The PAXgene blood RNA system and manual provide a troubleshooting guide for system problems, it should be noted that the troubleshooting guide may help solve any problems that may occur. Regarding low RNA yields, the troubleshooting guidelines indicate that: "less than 2.5ml of blood was collected in PAXgene blood RNA tubes. Ensure that 2.5ml of blood was collected in the PAXgene blood RNA tube (see PAXgene blood RNA tube product through-letter). Admittedly, the PAXgene blood RNA system is not designed for or successfully applied to small volumes of samples.

Comparing the PAXgene blood RNA system procedure and RNA isolation method with the methods provided herein (including example 1) will demonstrate a significant reduction in the volume used (including specifically the volume used in each step) and of about 20% of the indicated volume. The recommended and most suitable sample size for Paxgene systems is about 1/10 or 10% by volume, and can be treated with about 25% by volume of buffer and solution to successfully provide sufficient quality and quantity of RNA for whole transcriptome analysis and transcriptome spectroscopy by RNA sequencing.

According to one embodiment of the method, the volume of buffer (water) in step 2 is 1ml, which is 25% of 4ml in the kit method, compared to the volume of commercial RNA isolation kit, as in particular the PAXgene blood RNA kit method and procedure outlined above. According to one embodiment of the method, the buffer volume in step 4. Is 75. Mu.l, which is 21.4% of 350. Mu.l in the kit method. According to one embodiment of the method, the buffer volumes in step 5. Are 65. Mu.l buffer and 9. Mu.l proteinase K, which is 21.7% of 300. Mu.l and 22.5% of 40. Mu.l in the kit method. According to one embodiment of the method, the buffer volume in step 8. Is 75. Mu.l, which is 21.4% of 350. Mu.l in the kit method. According to one embodiment of the method, the buffer volume in step 11. Is 100. Mu.l, which is 28.5% of 350. Mu.l in the kit method. According to one embodiment of the method, the buffer volume in step 14. Is 100. Mu.l, which is 28.5% of 350. Mu.l in the kit method. The volume adjustment of the buffer and solution in the present method ranges from about 21% to about 29% or generally about 25%.

Robison and colleagues have previously reported an overall assessment of transcript profiling of finger prick blood samples (Robison EH et al (2009) BMC Genomics 10:617, doi:10.1186/1471-2164-10-617). Only the extensive correlation of RNA quality and gene expression data of finger stick samples with whole blood samples was reported using gene chip analysis. Robison follows the PAXgene blood RNA kit (product # 762164) protocol for RNA isolation and purification except for one modification, in which after the first rotation, the pellet was washed with 1mL of RNase-free water instead of 4mL because of its small volume. Robison reported that they tested a scaled-down version of the PAXgene protocol, but found that the use of standard volumes of buffer and wash had no effect on yield and was preferred for easier use. This is in sharp contrast to the studies and results reported and provided herein.

The methods herein may further comprise sequencing the RNA. The RNA can be sequenced using any suitable or recognized method, procedure, system, or kit, including manual, semi-automated, or automated methods, systems, or kits. In some embodiments, a kit (e.g., illumina TruSeq or Kapa Hyper Prep kit) is used.

The isolated RNA can be converted to cDNA as part of or commensurate with the methods herein. Methods for generating cDNA from RNA are well known and available to those skilled in the art. Any suitable and effective method should be suitable for converting isolated RNA into cDNA for probing of probes or specific primer applications, such as assessing expression or sequencing of specific RNA or gene products. The isolated RNA may be converted to cDNA for cloning purposes, inserted into or prepared in a vector, for introduction into or preparation of a library therefrom.

The isolated RNA can be amplified as part of or commensurate with the methods herein. In some embodiments, the RNA can be converted to cDNA and then amplified. Suitable amplification methods and systems are known and available. For example, methods, kits and systems for PCR amplification, including RT-PCR, in which RNA is first reverse transcribed to cDNA and then amplified, are well known and available amplification methods and pathways may be particularly useful in cases of small volumes of samples and/or isolation of small amounts of RNA. Another useful amplification pathway for small volumes or volumes of RNA samples is loop-mediated isothermal amplification (LAMP). RNA can be detected and assessed by combining LAMP with the reverse transcription step. LAMP is performed at a constant temperature (60-65 ℃) and therefore does not require a thermal cycler. The LAMP method may use Bst (Bacillus stearothermophilus ) DNA polymerase.

Abundant RNA species or RNA species not of interest may be removed prior to RNA sequencing. For example, globin mRNA, ribosomal RNA, and/or species-specific RNA may be removed prior to sequencing. In some cases, both the globulin and ribosomal RNAs are removed. This serves to eliminate highly prevalent RNA or known RNA of no interest from the isolated RNA. Elimination of highly prevalent or unrelated globulin RNAs or rrnas may aid in analysis of RNAs of interest or less prevalent and present in smaller amounts. Methods, systems and kits for removing globulin and/or ribosomal RNA are well known and available to those of skill in the art. In some embodiments, a system or kit may be used, such as Blobinzero (Yingda), ribo-Zero Gold, truSeq Total RNA library preparation, ribo-Zero Global in, GLOBINclear kit (Semer Fielder), QIAseqFastSelect RNA removal kit (Kanji). In some embodiments, species-specific probes may be used to select certain RNAs.

In embodiments or methods, the patient or individual has or is at risk of having or is suspected of having a disease or infection. The disease may be an acute or chronic disease. The disease may be a recurrent and/or remitting disease. The infection may be a bacterial or viral infection. Infection may be caused by known or unknown viral or bacterial masses. The viral infection or virus may be influenza virus, coronavirus, unidentified virus, RNA virus. The bacteria may be gram positive bacteria. The bacteria may be Streptococcus or Staphylococcus. The disease may be an inflammatory disease, an immune disease, an autoimmune disease, a cancer.

In some embodiments, the method is for longitudinal screening by RNA profiling and analysis of a small volume sample from one or more patients or individuals who have or are at risk of or suspected of having a disease or infection. In embodiments, small volumes of sample are collected continuously or regularly or in specified increments of hours, days, weeks, or months. A small volume of blood sample, sputum or saliva sample, or a small volume of a nasal, nasopharyngeal or oropharyngeal swab, wash or aspirate may be collected. A combination of sample types or different sample types may be collected. In embodiments, small volumes of sample are collected continuously or regularly by finger prick or in specified increments of hours, days, weeks or months.

The sample may be collected in increments of several hours, twice a day, three times a day, every 4-6 hours, daily morning, daily evening, daily morning and evening, weekly, monthly, every two months, every four months, every six months, multiple times a year. Samples may be collected to assess the effect of the drug or agent, for example, before and/or after administration of the drug or agent. In some embodiments, a small volume sample may be collected or otherwise collected at the onset of symptoms (e.g., one or more symptoms or a recognized indicative parameter associated with a disease or associated with an infection). The sample may be collected before and after the identification or development of one or more symptoms or disease or infection parameters or in accordance with such symptoms or disease or infection parameters. Samples may be collected as fever, cough, pain or discomfort, rash develop.

Systems and kits for using and applying these methods are provided. A system or kit for RNA profiling and analysis of a small volume sample from a patient or individual is provided, comprising:

(a) Means for collecting small volumes of sample by the patient or individual or non-medical personnel, including lancets, swabs or containers for washing, spitting or aspiration;

(b) A tube or container for receiving the small volume of sample at the time of collection and containing a volume of RNA stabilization solution, whereby cells in the sample are lysed and RNA is stabilized; and

In one embodiment, with collection, the first drop of blood is removed, such as with sterile gauze or cotton balls, to avoid interstitial fluid that may produce inaccurate or less effective results. In one embodiment, the finger, heel, etc. is cleaned, wiped or swabbed with an alcohol or detergent solution prior to collection to remove any surface debris, loose cells or bacteria or dirt.

In some embodiments, the lancet can be a small manual blade or can be a spring-loaded assembly or a self-contained disposable unit, for example, where the blade is automatically retracted into the holder after use. One such example is a Dynarex SensiLance pressure activated lancet.

(a) A set of multiple means for collecting small volumes of sample by a patient or individual or by non-medical personnel, each set comprising a lancet, swab or container for washing, spitting or aspiration;

(b) A set of a plurality of tubes or containers, each tube or container individually for receiving a small volume of the collected sample and containing a volume of the RNA stabilizing solution such that cells in the sample are lysed and the RNA is stabilized;

(c) A variety of suitable labels are used to designate the name or identity of the patient or individual, the date of sample collection, and the time of sample collection; and

(d) Various envelopes and mailing containers are used to transport each sample or multiple samples to a laboratory or facility for RNA isolation and analysis.

In some embodiments described in the system or kit, the volume of the RNA stabilizing solution is less than 1ml, about 500. Mu.l or less, about 300. Mu.l or less, about 200-300. Mu.l, or about 250. Mu.l.

In some embodiments described in the systems or kits, the tube or container for receiving a small volume of sample and containing the RNA stabilization solution has a total volume capacity of 1.5ml or less, 1.2ml or less, or 1ml or less.

In the description, numerous specific details are set forth in order to provide a thorough understanding of the present embodiments. However, it will be apparent to one skilled in the art that the present embodiments may be practiced without the specific details. In other instances, well-known materials or methods have not been described in detail in order to avoid obscuring the present embodiments.

Throughout the specification, amounts are defined by ranges and the lower and upper limits of the ranges. Each lower limit may be combined with each upper limit to define a range. The lower limit and the upper limit should be taken as separate elements, respectively.

Furthermore, any examples or descriptions given herein should not be taken as limiting, restricting, or defining explicitly the use of any term in any way. Rather, these examples or illustrations should be considered as being described with respect to one particular embodiment and are merely illustrative. Those of ordinary skill in the art will understand that any one or more terms used with these examples or descriptions will encompass other embodiments, which may or may not be presented elsewhere herein or in the specification, and that all such embodiments are intended to be included within the scope of the term or between terms. Language specifying such non-limiting examples and illustrations includes, but is not limited to: "for example," such as, "" as, "and" in one embodiment.

In this specification, various parameter sets including a plurality of members are described. Each member may be combined with any one or more of the other members in a set of parameters to form additional subgroups. For example, if the members of one group are a, b, c, d and e, then other subgroups specifically contemplated include any one, two, three, or four members, e.g., a and c; a. d and e; b. c, d and e; etc.

The invention will be better understood with reference to the following non-limiting examples, which are provided as examples of the invention. The following examples are provided to more fully illustrate the preferred embodiments of the invention, but should in no way be construed as limiting the broad scope of the invention.

Example 1

Isolation and analysis of RNA from finger prick blood samples from rheumatoid arthritis patients: RNA-based longitudinal genome Marker for identifying RA attack

Similar to many inflammatory diseases, rheumatoid Arthritis (RA) is characterized by the occurrence of stationary and exacerbating (seizure) leading to seizure molecular events that are not yet known. We have established methods, kits and systems, as well as clinical and technical protocols, for RNA isolation by repeated home blood collection of RA-patients via self-finger-stick sampling of small volume blood samples. The system enables and allows for longitudinal RNA sequencing (RNAseq). Samples were taken from 364 time points of our index patients with eight episodes in four years, and 235 time points of the episodes of the other three patients. We have developed a sampling method and RNA stabilization and isolation protocol that provides high quality intact RNA. The evaluation results show that RNAseq data from small volume blood finger stick samples correlates with blood cell count of venipuncture blood draw. We determined transcripts that were differentially expressed prior to onset and compared them to synovial single cells RNAseq (scRNAseq). Flow cytometry and sorted blood cell RNAseq were used in other RA patients to verify the findings.

One to two weeks prior to RA onset, consistent changes were observed in blood transcriptional profile (profiles). B cell activation is followed by expansion of previously unexplored circulating CD 45/CD 31/pdpn+, pre-inflammatory mesenchymal ("PRIME") cells in the blood of RA patients, which share the characteristics of inflammatory synovial fibroblasts. During the onset of all four patients, circulating PRIME cells were depleted, and flow cytometry and sorted cellular RNAseq confirmed the presence of PRIME cells in the other 19 RA patients.

Longitudinal genomic analysis of RA episodes revealed PRIME cells in RA blood and proposed a model in which they were activated by B cells a few weeks before RA episodes and then migrate outward from the blood to the synovium. These studies indicate that the collection system and RNA stabilization and isolation methods allow RNA sampling to obtain valuable and consistent results. In various applications of the system and method, RNA profiling and longitudinal RNAseq analysis using the system and method may reveal dynamic changes leading to the onset of chronic inflammatory disease.

Introduction to the invention

Symptoms of Rheumatoid Arthritis (RA) are highly dynamic, with the stationary phase being interrupted by unpredictable episodes of disease activity. The clinical course of such fluctuations is a feature of many autoimmune diseases, including multiple sclerosis (1), systemic lupus erythematosus (2) and inflammatory bowel disease (3, 4), emphasizing the need to develop methods to understand what triggered the transition from resting state to onset of autoimmune disease.

The study explored disease pathophysiology by taking small volumes of blood samples from patients by finger stick at home, and performing a longitudinal, prospective analysis of blood transcriptional profile characteristics over time in individual RA patients. Previous microarray studies on RA blood samples (from relatively sparse time series data) have hardly identified significant genetic changes associated with disease activity (5-8). Here we provide a first RA study to find molecular changes in blood that are predictive of clinical onset. For this reason, we developed and optimized methods that allow RA patients to collect high quality finger prick blood samples for RNA sequencing (RNAseq) themselves, facilitating blood collection for months to years per week.

We analyzed patient reports from 4 patients of clinical disease activity and RNAseq data across multiple clinical episodes. In our most deeply studied index case, we assessed 364 time points for 8 episodes in 4 years by RAPID3 and analyzed 84 time points for RNAseq assessment. Longitudinally collecting the sample allows us to find transcriptional signatures prior to clinical symptoms. Comparison of these blood RNA profile features with synovial single cell RNAseq (scRNAseq) data (9) provides evidence that a set of biologically relevant transcripts in the blood significantly increases before symptoms appear and that a subset decreases as the patient begins to appear. These latter transcripts overlap and possibly are distinguishable from cell precursors with the new subset of synovial lower fibroblast types detected in inflamed RA synovium using scRNAseq. Analysis of an additional 19 RA patients confirmed our findings. Our data indicate a model in which previously unexplored circulating mesenchymal cell types, detectable a few weeks before RA onset, become activated by B cells, then leave the blood, enter the synovium, and cause disease activity. The ability to isolate and analyze high quality RNAs that effectively represent in vivo changes in small volume blood samples (referred to as lancing blood samples) collected by patients themselves has facilitated these studies.

Method

Patient data

All patients met the RA standard of the american college of rheumatology/european anti-rheumatosis alliance 2010 (10, 11) and cyclic citrullinated protein antibody (CCP) serum responses were positive. The disease activity is assessed weekly in the home, or up to 4 times per day during the course of the seizure escalation, using routine assessment of the patient index data 3 (RAPID 3) questionnaire (12). RAPID3 and disease activity score 28 (DAS 28) were also used to assess disease activity during outpatient, monthly and seizure periods, which incorporates a comprehensive assessment of tenderness and swelling, erythrocyte Sedimentation Rate (ESR) and patient disease activity from 28 joints. Whole blood count (CBC) including White Blood Cells (WBC), neutrophils, monocytes, lymphocytes and platelets was performed by the clinical laboratory of the commemorative Stonex-Kailin cancer center. We collected 43 outpatients indexing patients, and 25, 14 and 12 outpatients of the other three patients, for longitudinal study. By FACS and RNAseq analysis, the presence or absence of PRIME cells in another 19 seropositive RA patients and 18 age and sex matched non-RA patients available to Peripheral Blood Mononuclear Cells (PBMCs) was also studied.

Preparation of RNA from finger lancing

The patient performed finger prick at his own home to collect three drops of blood into a microtainer tube pre-loaded with fixative (RNA stabilization solution), and the samples were mailed overnight weekly. RNA was extracted using the PAXgene RNA kit and purified according to the manufacturer's protocol, except that the volumes of all washes and elutions were reduced to about 25% of the manufacturer's recommended volume. The amount and quality of RNA was evaluated using an Agilent bioanalyzer (Agilent bioanalyzer). For library preparation we used the GlobinZero kit (EpiCentre #gzg1224) and the Truseq mRNA chain library kit of Illumina, 5-8nM input using 11-12 PCR cycles and sequencing on HiSeq2500 with 150 base pairing end reads. The readings were aligned with Gencodeev 18 using STAR and quantified using a featuremake (v1.5.0-p 2). At least four million paired-end reads of the sample were retained for analysis.

The following provides detailed solutions:

manipulation (SOP) finger prick sample handling

Should receive a box containing a patient questionnaire and finger stick sample

Thawing and drying ice bags in a cardboard box lined with a chuck

Notes are taken in the sample record:

date of sample collection (date of lancing blood) date on questionnaire

Date of the day

Study week # (if the previous week was not clear, please calculate starting at week 0 at the top of the sample recording page)Finger prick sample Product(s)

Three drops of blood were received in a microtainer tube pre-filled with 0.250ml (250 μl) of PAX-gene blood solution.

Writing (finger-pricked) date on finger-pricked label

Transfer to-20 ℃ for 24 hours. Is vertically stored in the metal rack.

Transfer to-80 ℃ for long-term storage in-80 ℃ patient sample cartridges, and write cycle # on top of the tube.

(adapted from PAXgene RNA Manual 2 nd edition, month 6 2015)

Before starting

The temperature of the shaking incubator was set to 55 DEG C

If there is a precipitate, buffer BR2 (binding buffer) is heated to 37℃and the binding buffer contains a guanidine salt (guanidine thiocyanate) which forms a highly active compound when combined with the bleaching agent

Buffer BR4 (washing buffer) was prepared by adding 4 volumes of 100% ethanol to obtain working solution

DNase I stock was prepared by dissolving solid DNase I (1500 Kunitz units; qiagen, catalog number 79254) in 550ul of RNase-free water and mixing upside down (1500 Kuntz units/0.55 ml) without vortexing, DNase being sensitive to physical denaturation.

1. PAXgene blood RNA microtainer blood collection tube was removed from the refrigerator and allowed to warm to room temperature (+/-1 hour)

2. PAX blood was placed in a 2ml microcentrifuge tube and centrifuged at 5000x g for 10 minutes at room temperature

3. The supernatant was removed and discarded and 1ml of RNase-free water (from the PAXgene kit) was added to wash the pellet.

4. Vortex to resuspend pellet and then centrifuge in a centrifuge at 5000x g for 10 minutes. Remove and discard supernatant

5. The pellet was thoroughly resuspended in 75ul buffer BR1 (from PAXgene kit) by vortexing (resuspension buffer)

6. 65ul of buffer BR2 (from PAXgene kit) and 9ul of proteinase K solution (from PAXgene kit) were added.

7. Mix by vortexing and incubate in a shaking heating module (800 rpm) at 55 ℃ for 10 minutes.

8. The lysate was pipetted onto a PAXgene crushing centrifuge column (which can remove the clumps) on top of the light purple and spun at 18,000xg for 3 minutes.

9. The transfer of the supernatant of the effluent (about 150 ul) to a new 1.5ml microcentrifuge tube is careful because the pellet is viscous and easily broken

10. 75ul of 100% ethanol was added. The droplets in the cover tube were removed by vortex mixing and centrifugation at 1000x g for 2 seconds. Centrifugation should not take place beyond this time, otherwise nucleic acids may precipitate and decrease RNA yield.

11. 225ul of sample was applied to a PAXgene RNA centrifugation column placed on top of red in a 2ml processing tube (from PAXgene kit). 8000 and x g for 1 minute. The PAXgene column was placed in a new 2ml processing tube and the old processing tube containing the effluent was discarded.

12. 100ul of buffer BR3 (wash buffer) was pipetted into the PAXgene column and centrifuged at 8000x g for 1 min. The PAXgene column was placed in a new 2ml processing tube and the old processing tube containing the effluent was discarded.

13. 5ul DNAA enzyme I stock was pipetted into 35ul buffer RDD. Mix by gently shaking the tube (without vortexing) and briefly centrifuge.

14. The DNase I incubation mixture (40 ul) was directly pipetted onto a PAXgene column and placed upright for 15 minutes at room temperature.

15. 100ul of buffer BR3 was pipetted into the PAXgene column and centrifuged at 8000x g for 1 min. The PAXgene column was placed in a new 2ml processing tube and the old processing tube containing the effluent was discarded.

16. 200ul of buffer BR4 (wash buffer) was transferred to the PAXgene column and centrifuged at 8000x g for 1 min. The PAXgene column was placed in a new 2ml processing tube and the old processing tube containing the effluent was discarded. It is noted that buffer BR4 is provided as a concentrate. Ensure that ethanol is added to buffer BR4 prior to use.

17. An additional 200ul of buffer BR4 was added to the PAXgene column. Centrifuge at 18000x g (maximum speed) for 3 minutes to dry the PAXgene column membranes.

18. To remove residual buffer BR4, the effluent containing tube was discarded, and the PAXgene column was placed in a 2ml processing tube and centrifuged at full speed for 1 min.

19. The tube containing the effluent was discarded and the PAXgene column transferred to a 1.5ml elution tube (kit from PAXgene). 30ul of buffer BR5 was pipetted directly onto the PAXgene column membrane (pipette tips do not touch the membrane) and centrifuged at 13000x g for 2 min.

20. The elution step was repeated as described using 30ul of RNA previously eluted in buffer BR5 (elution buffer). 2ul for Pico bioanalyzer, 26ul for Globinzero

21. Samples were labeled and stored at-80 ℃ until RNA analysis was performed.

Data analysis:

differential expression analysis across patients

Samples were marked as "baseline" (stabilized RAPID 3), "seizure" (RAPID 3 score increased by two standard deviations over baseline mean) or "steroid". Edge r (v3.24.3) (13) was used to analyze differential gene expression of episodes relative to baseline. Substitution test (n=1x106) was used to test the significance of overlap between the genes reduced at the onset in index patients and patients 2, 3 and 4. GO enrichment (goana, from limma v 3.38.3) (14) was used to identify an enrichment pathway for significantly differentially expressed genes in index patients (FDR < 0.1) and the direction of expression was consistent for index and repeat patients (i.e., log fold changes were either positive or negative).

Indexing time series analysis of patients

We performed longitudinal data analysis on index patients using ImpulseDE2 (v1.8.0) (15). Onset of onset was clinically defined (as described above), samples from 8 weeks before onset to 4 weeks after onset were analyzed (excluding any samples during steroid administration by the patient, n=65 samples). Library preparation date inclusion model batch corrections were performed and the genes under expressed were screened using the genefile (v 1.64.0) package (16). We hierarchically clustered the average expression of significantly differentially expressed genes by week until onset of onset (batch corrected logrpkm expression values were calculated using edge) and determined five co-expressed gene modules (clusters 1-5). We analyzed the GO term enrichment (goana) of these five modules.

To compare the differentially expressed gene modules over time, the average expression level of each gene at weekly episodes was calculated and then weekly normalized. ABIS (17) and CIBERSORTx (18) are used to deconvolute gene expression data. To aggregate a given gene cluster or cell type with gene markers, the average of the normalized gene expression fraction or deconvolved cell type fraction per week is plotted, respectively. To identify synovial scRNAseq cluster-specific marker gene signatures, we compared cells from one scRNAseq cluster to cells from all other scRNAseq clusters using a single cell RNA-seq log2 (cpm+1) matrix using the previously published dataset (18). We generated a list of the first 200 marker genes for each cluster using 1) a criteria of log2FC greater than 1, 2) auc greater than 0.6, and 3) a percentage of expressed cells greater than 0.4. We used the fischer accurate test to assess the degree of enrichment of synovial cell subtype marker genes in 5 co-expressed gene modules.

Flow cytometry and sorting

Samples from PBMCs were stained with the following antibodies: CD31-APC, (WM 59), mouse IgG1-APC, (MOPC-21), PDPN-PerCP, (NZ 1.3), rat IgG2a, (eBR 2 a), CD45-PE, (HI 30), mouse IgG1-PE, (MOPC-21), mouse IgG2a,-3 and DAPI (4', 6-diamidine-2-phenylindole, dihydrochloride). Cells were sorted on BD FACSAria II for RNAseq. The cDNA library was sequenced on MiSeq. DESeq2 (v1.24.0) (19) was used for differential expression analysis.

Statistics of

R2 and pearson correlation coefficients were calculated to evaluate bivariate linear fits of disease activity measured by RAPID3 and DAS28 and CBC counts inferred from cibert cell counts and counts measured by clinical laboratories. The deduced cibelsortx lymphocyte count is the sum of naive B cells + memory B cells + CD 8T cells + CD4 naive T cells + CD4 memory resting T cells + CD4 memory activating T cells. One-way ANOVA was used to test for significant differences between various clinical features according to disease activity status. Monocytes, macrophage M0, macrophage M1 and macrophage M2 were summed to infer cibelsortx monocytes.

Results

Development of clinical protocol

Four RA patients were followed for one to four years, finger prick blood samples were collected weekly in the home, and RAPID3 and monthly clinic were completed with DAS28 collected (fig. 1A). Study patients also recorded disease activity (RAPID 3 questionnaire). We developed a strategy for home blood collection that could provide high quality and high amounts of RNA for sequencing (FIGS. 2-8), which provided 15-50ng RNA and RNA Integrity (RIN) scores (average 6.9+/-standard deviation 1.7) from finger prick blood samples.

Total 189 finger prick blood samples from 4 patients were sequenced for RNA extraction, of which 162 (87%) passed the quality control screen.

We first assessed the quality and quantity of RNA by fixative volume. 3 drops of blood collected with a 21-gauge needle were added to a microtainer blood collection tube preloaded with 250. Mu.l, 500. Mu.l, or 750. Mu.l of PAX gene fixative. Samples were stored at room temperature for 3 days, then RNA was extracted using a PAX gene RNA kit, and RIN scores and the amount of RNA were assessed using an Agilent 2100 bioanalyzer picochip. RIN denotes the number of RNA integrity, which is an algorithm for evaluating RNA integrity values. The integrity of RNA is of great importance for gene expression studies. RIN can and traditionally is evaluated using a 28S (about 5070 nucleotides) to 18S (about 1869 nucleotides) RNA ratio of about 2.7. A high ratio of 28S to 18S indicates that the purified RNA is intact and not degraded. The RIN (Schroeder A et al (2006) BMC Mol Biol 7:3 (doi: 10.1186/1471-2199-7-3). RIN score of RNA samples should be >7 in the range of 1 (highly degraded) to 10 (highest integrity) was easily determined using Agilent 2100 bioanalyzer measurements the results are shown in FIG. 2. Acceptable RIN score can be seen with 250. Mu.l, 500. Mu.l or 750. Mu.l PAX-gene fixative (left panel of FIG. 2.) it is noted that 250. Mu.l fixative results in highest ng RNA yield per sample using higher volumes of fixative, whether 500. Mu.l or 750. Mu.l fixative, the ng RNA yield was significantly reduced compared to 250. Mu.l fixative (right panel of FIG. 2).

RNA integrity/quality and RNA quantity were assessed from 100. Mu.l blood samples in 250. Mu.l PAX gene fixative, which were stored at room temperature for various times (FIG. 3). 100 μ1 whole blood was added to a microtainer tube pre-loaded with 250 μ1PAX gene fixative and frozen after incubation for 2 hours, 3 days or 7 days at room temperature. RNA was extracted using the protocol described above using a downscaled wash and elution, and the RIN score and number of RNA was estimated using an Agilent 2100 bioanalyzer RNA picochip. The RNA quality and quantity are reasonably preserved after 3 days of storage at room temperature.

RNA quality and quantity were assessed in fresh and mailed samples (fig. 4). 100 μ1 whole blood was added to a microtainer tube pre-loaded with 250 μ1PAX gene fixative and incubated at room temperature for 2 hours or post-mailed frozen. RNA was extracted as described above and RIN scores and amounts of RNA were assessed using an Agilent 2100 bioanalyzer RNA picochip. By mailing the sample, the amounts of RIN and RNA remained good.

The quality and quantity of RNA was assessed in terms of extraction and washing volumes (FIG. 5). 3 drops of blood collected with a 21-gauge needle were added to a microtainer tube previously filled with 250. Mu.l of PAX gene fixative. Samples were stored at room temperature for 3 days, then RNA was extracted using a PAXgeneRNA kit or PAX protocol in a downscaled form according to the manufacturer's instructions, all washes and elutions were performed using a significantly reduced volume (about 25% of the recommended volume). RIN score and RNA quantity were assessed using an Agilent 2100 bioanalyzer RNA picochip. The RIN score remained good in the low volume regimen. However, the amount of RNA isolated by the low volume protocol is significantly improved. This suggests that a volume reduction protocol is necessary in order to separate a reasonable amount of RNA from a small blood volume sample (e.g., a few drops of blood corresponding to the size of a finger stick type blood sample).

RNA was isolated using PAXgeneRNA extraction versus TriZol-based methods, and RNA quality and quantity was assessed from finger prick blood samples. The mailed patient finger stick samples were stored in PAXgeneRNA buffer at-80 ℃.142 samples were RNA extracted by PAXgeneRNA extraction and low volume washed while 13 samples were thawed and mixed with 700. Mu.l Trizol-LS and 250. Mu.l chloroform. After centrifugation, the top layer was precipitated with isopropanol and glycogen, washed with 80% cold ethanol, centrifuged and the precipitate dried, resuspended in PBS and then purified using Luo Shigao pure separation kit. The integrity and quality of RNA extracted using Trizol and chloroform was significantly reduced compared to the PAXgene RNA system. Trizol reagent system uses guanidine thiocyanate and phenol, and organic extraction is performed by phenol/chloroform.

Since ribosomal and hemoglobin RNAs represent about 98% and 70% of RNA in whole blood, respectively, we tested standard commercial kits for removing these RNAs prior to RNAseq. The PAXgene system does not remove the globin mRNA, which accounts for 70% of the mass of mRNA in the total RNA of whole blood. GlobinZero (Illumina) methods and kits are used to remove globin mRNA from a sample. 4ml of heparinized blood was treated with 1ug/ml LPS at 37℃for 1 hour and 250ul of blood was placed in 250ul of PAXgene fixative in duplicate microtainer blood collection tubes. After RNA extraction, the samples were treated with a globulin zero-depletion kit (globulin or ribosome depleted) or not, and then quantitative PCR was performed to detect the expression of hemoglobin A2, 18SRNA or tnfα mRNA. FIG. 7 depicts the cycle time of HbgA2, 18S RNA and TNF. Alpha. After Globinzero depletion. The globinlzero kit depletes hemoglobin A2 and 18S ribosomal RNAs (average cycle time increases from 11 to 28 and 10 to 30, respectively) while retaining tnfα mRNA.

RNASeq QC index was evaluated on RNA prepared using Illumina TruSeq or Kapa Hyper Prep kit, and RIN score ranged from <5.7 to 8.1-10 (FIG. 8). Mapping distributions, unique mappings and duplicate readings of TruSeq and Kapa Hyper Prep RNA with different RIN scores are plotted. The tag distribution of UTR (untranslated region), intergenic region, intron, and CDS (coding sequence) assigned to whole blood RNA samples prepared with Illumina TruSeq or Kapa Hyper Prep kit with various input RNA qualities and amounts was determined. Illumina TruSeq library preparation showed increased mapping of coding sequences and fewer intergenic region reads and was ultimately used in downstream experiments.

To assess the effectiveness of patient-reported disease activity, we compared their RAPID3 score to the DAS28 collected by the clinician. There was a significant correlation between RAPID3 and DAS28 for four patients (fig. 1B). To assess the effectiveness of finger prick blood data, we compared RNAseq inferred white blood cell count to clinical laboratory measured whole blood count and again observed significant correlation (fig. 1C). Taken together, these data indicate that patient reports of disease activity are paired with finger prick blood samples, providing a high quality and powerful means for individuals to participate in longitudinal clinical studies.

RA episode compared to baselineClinical and molecular characterization of (C)

Attacks are associated with an increase in objective clinical and laboratory measurements that index RA-related disease activity in patients (fig. 9A). Finger prick RNAseq determined differential expression of 2613 genes relative to baseline at onset (FDR < 0.1), with 1437 increasing during onset (logFC >0; fig. 9B). Pathway analysis determined enrichment of bone marrow, neutrophils, fc receptor signaling, and platelet activation (fig. 9C), consistent with clinical CBC measurements during the seizure. Interestingly, 1176 genes were significantly reduced during the seizure period, and the pathway analysis of these genes was enriched in extracellular matrix, collagen and connective tissue development (fig. 9D).

Time series analysis of molecular events leading to RA episodes

To analyze the trajectory of gene expression over time and determine the potential pre-cause of the seizure, we performed a time series analysis of RNAseq data (fig. 10A) notably that the disease activity score for several weeks prior to seizure was the same as the baseline score for two months prior to seizure, underscores the challenges of identifying the time frame and gene expression profile prior to seizure. We focused on analysis of 65 samples collected 8 weeks before and 4 weeks after onset of the seizure, classifying the samples according to the number of weeks the samples were drawn. This determined that 2791 genes had significantly different expression over time to onset (FDR < 0.05), and hierarchical clustering of gene expression determined five clusters (fig. 10B). Cluster 1 represents a group of genes that increased after symptoms appeared (fig. 10C and 10D) and highly overlapped with genes that increased in seizure versus baseline analysis (fig. 9B) (fig. 10E). These gene expression clusters were reproducibly altered in 5 independent clinical seizure events (fig. 12).

We further focused on the developmental pathways of primary B cells and leukocytes that increased two weeks prior to onset due to cluster 2 (AC 2) (table 2) transcripts differentially expressed prior to two clusters prior to onset (fig. 10C-10D). The other two methods of deconvolution of RNAseq data, CIBERSORTx and ABIS, independently confirmed evidence of prior onset of B-cell and T-cell populations, and all analyses showed evidence of innate inflammatory features (neutrophils and monocytes) during onset (data not shown).

The pre-cause cluster 3 (AC 3) (table 3) transcripts increased one week prior to the episode and then decreased during the episode (fig. 10C and 10D). Atypical pathways for AC3 enrichment in blood samples, including cartilage morphogenesis, cartilage in-bone growth, and extracellular matrix tissue (fig. 10E), indicate the presence of uncharacterised cell types.

Time series analysis of synovial cell marker genes in RA episodes

To better characterize the correlation of clusters determined by time series analysis with synovitis (fig. 10C), we examined their enrichment in the scRNAseq-characterized synoviocyte subtypes. Analysis of 5265 individual RA and synovial cells from osteoarthritis patients determined four fibroblasts, four B cells, six T cells and four monocyte subpopulations (fig. 11A). We determined about 200 marker genes that best differentiated 18 synovial cell types. AC2 was enriched with naive B cell genes (fig. 11A), AC3 was enriched with three lower fibroblast genes (cd34+, HLA-dr+ and dkk3+) (fig. 11A). Two of the fibroblast subpopulations, CD34+ and HLA-DR+, were more abundant in inflamed synovium (20). We plotted the expression of transcripts common to the sub-synovial fibroblasts and AC3 over time and again noted their increased expression in blood one week prior to onset, while decreased expression during onset (fig. 11B and table 1).

Overall, 622 of 625 AC3 genes decreased during the episodes of patient 1, and a subset (194 genes) also decreased in the episodes of at least 3 of 4 RA patients (4 of the 22 genes in 4 patients; fig. 11C), and substitution testing indicated that this overlap was fortuitously greater than expected (p=0.0001). Pathway analysis of 194 overlapping subsets of genes again enriches extracellular matrix and secreted glycoproteins.

We further tested by flow cytometry whether cells expressing synovial fibroblast surface markers could be detected in RA blood. CD45-/CD31-/PDPN+ cells were increased in the blood of another 19 RA patients relative to healthy controls (FIG. 11D). RNAseq of these cells demonstrated that they were rich in AC3 cluster genes (fig. 11E), synovial fibroblast genes (fig. 13), and expressed classical synovial fibroblast genes (e.g. FAP, DKK3, CDH 11) collagen and laminin (fig. 14). Considering their expression of classical mesenchymal surface markers and genes, we refer to them as pre-inflammatory mesenchymal cells (PRIME cells). Taken together, our observations suggest a model in which continuous activation of B cells activates PRIME cells immediately prior to onset, and then becomes apparent at onset as inflammatory lower fibroblasts in the inflamed synovium.

Reference table 1 is as follows:

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discussion of the invention

We have considered longitudinal genomics as a strategy to study the pre-cause of RA episodes that can be generalized to autoimmune diseases associated with fluctuating clinical courses. Over the years, we have developed easy-to-use tools for patients to acquire quantifiable clinical symptoms and molecular data at home. This enabled us to capture data before clinical onset and to retrospectively analyze it, identifying distinct RNA features (AC 2 (table 2) and AC3 (table 3) in peripheral blood 1-2 weeks before onset.

The RNA profile of AC3 and sorted CD45-/CD31-/PDPN+ circulating cells revealed enrichment of pathways including cartilage morphogenesis, endochondral bone growth and extracellular matrix tissue (FIG. 10E) and a strong overlap with synovial lower layer fibroblasts. Thus, we propose that pre-PRIME cells are precursors to inflammatory lower fibroblasts, which were previously found near blood vessels in the inflamed RA synovium (21).

Importantly, inflamed underlying fibroblasts are pathogenic in an animal model of arthritis (22). We found that the human AC3 gene has the molecular characteristics of underlying fibroblasts and that these cells were observed to peak before but not readily detected in the blood during the onset (fig. 9 and 10), supporting a model in which PRIME cells migrate acutely from the blood to the synovium where they promote the inflammatory process. This model is consistent with the observation that RA synovial fibroblasts can be transported to cartilage implants and are sufficient to passively transfer synovial inflammation (in mice) (23). Our data together indicate that the mesenchymal signal detected in AC3 prior to onset represents a previously uncharacterized type of transport fibroblast cells circulating in the blood.

Furthermore, we observed that the second RNA feature, AC2, was activated in the blood before the AC3 peak. AC2 has the RNA characteristics of naive B cells. This finding reminds a recent study, demonstrating that autoreactive naive B cells are specifically activated in RA patients (24). Although these triggers are unknown, infectious (e.g., bacterial or viral antigens), environmental or endogenous toxins (25-27) can provide a source of specific antigens or activation pattern recognition receptors.

In summary, we demonstrate a method for densely collecting longitudinal clinical and gene expression data that can be used to discover changes in transcriptional profile in blood several weeks prior to onset of symptoms. These methods include methods and procedures for stabilizing, isolating and analyzing RNA from small-volume samples that may be collected by a patient or individual themselves, such as by finger stick, without medical personnel, and are suitable for home or field collection, for patients who are impaired or unable to collect blood by venipuncture, and for patients who require rapid sampling or periodic sampling over time. This approach led to the identification and characterization of RNA markers and disease or pathological condition indicators, and PRIME cells with synovial fibroblast characteristics were also found, which are more common in RA patients and increased in the blood prior to onset. In modeling all of our data, we propose that systemic B cell immune activation (detected as AC 2) acts on PRIME cells prior to clinical onset, these cells enter the blood (detected as AC 3) and subsequently enter the subsynovial layer during onset of disease activity.

More generally, the use of efficient self-collection protocols and methods in combination with quantitative and qualitative RNA isolation works in RA and RA patient sampling to demonstrate the effectiveness and utility of our system. This preliminary study provides an example of a method for isolating, assessing and evaluating markers and RNA or protein expression changes and cellular changes suitable for disease assessment and assessment, including fluctuations in inflammatory disease, suggesting a general strategy associated with a variety of diseases and conditions, including other disorders such as lupus, multiple sclerosis and vasculitis.

TABLE 2AC2 Gene

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TABLE 3AC3 Gene

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Example 2

The systems and methods detailed herein and in example 1 provide a framework and methods for assessing individuals at risk of disease, for assessing disease progression, or for identifying markers of more severe disease or metastasis. The availability of systems and methods for isolating and analyzing high quality RNA that can effectively recognize RNA, including in vivo RNA changes and new or altered RNA, such as RNA of different cellular components or infectious agents, from samples collected by the patient, including small sample volumes of blood samples (referred to as blood lancets), provides a means of monitoring and assessing various diseases or infectious diseases or conditions, particularly where traditional blood sampling is unnecessary, impractical or impractical. Applications for assessing and monitoring cancer patients are contemplated, including both before and after treatment or remission, as well as patients suffering from recurrent or remission diseases such as multiple sclerosis, crohn's disease, and the like. In addition, the systems and methods may be applied and practiced in infectious diseases, including viral diseases that affect a large population of people in seasonal or unexpected situations. Those at risk of infection or those suspected or identified to be infected can be evaluated to assess RNA indices of RNA response and disease, to characterize predictors or markers of susceptibility or disease severity, and to identify targets for treatment or modulation. For example, a more accurate and marker-based knowledge and understanding of susceptibility to influenza infection can reduce the impact of seasonal influenza on individuals and healthcare systems. In addition, the recent outbreak of the novel coronaviruses SARS-COV2 and COVID-19 pandemic underscores the urgent need for the systems, methods and approaches provided herein.

Coronaviruses are a type of virus that can cause diseases such as common cold, severe Acute Respiratory Syndrome (SARS), and Middle East Respiratory Syndrome (MERS). 3 months in 2020, world Health Organization (WHO)) announced an outbreak of covd-19 as a pandemic. By the beginning of 4 months, the number of globally confirmed cases of covd-19 was nearly 150 tens of thousands, with more than 400,000 in the united states, more than 135,000 in italy and spanish each, more than 100,000 in germany, and more than 80,000 in china reported. Worldwide, the number of deaths exceeds 80,000. No accepted or approved therapeutic methods or vaccines are available.

Signs and symptoms of covd-19 appear 2 to 14 days after exposure, and may include fever, cough, shortness of breath, or dyspnea, as well as tiredness, pain, runny nose, and sore throat. Some people lose their sense of smell or taste. Persons older or suffering from chronic diseases such as heart disease, lung disease or diabetes, or persons with impaired immune system may be at higher risk of suffering from severe diseases, similar to those in other respiratory diseases such as influenza.

The severity of the symptoms of covd-19 varies from very mild to severe, and some people may not have any symptoms at all. In fact, studies have shown that a significant portion of coronavirus infected individuals are asymptomatic ("asymptomatic"), even those who eventually develop symptoms ("pre-symptomatic") can transmit the virus to others before they develop symptoms (Li R et al Science 10.1126/Science. Abb3221 (2020); rotheC et al (2020) New Engl JMed 382 (10): 970-971; zou L et al (2010) New Engl J Med 382 (12) 1177-1179). Thus, viruses can spread between closely interacting people, e.g., speaking, coughing, or sneezing, even though these people do not exhibit symptoms.

According to the reports from disease prevention control centers, nearly one third of covd-19 disease cases in the united states are 6 years old or older, while patients over 65 years old account for nearly half of hospital treatments and the vast majority of deaths. Nonetheless, about 20% of 20-44 year old infected persons are hospitalized, indicating that this is not merely an elderly disease. There are significant and ongoing problems with regard to the potential biological vulnerability of the elderly and how preexisting conditions or diseases exacerbate covd-19. In addition, it would be helpful to provide an index for those patients who will develop more serious or more severe disease, so they can be managed or categorized differently or more aggressively.

RNA monitoring and longitudinal genomics according to the systems and methods provided herein, including as described in example 1, provides a method of isolating, identifying, and assessing RNA in a patient exposed to or at risk of a viral infection, such as a coronavirus infection, e.g., SARS-COV2 infection, or infection with the virus and diagnosed as covd-19. The system and method can be practiced in individuals after vaccination to assess RNA, protein, and cellular responses. The collection of blood samples in small amounts referred to herein may be collected at home, in a hospital or hospital, periodically by medical personnel or personnel, or in quarantine or quarantine. This allows monitoring of infection, including viral RNA, disease, RNA response, RNA alteration, including as an indicator of cellular response, as described above and in example 1. Obtaining high quality RNA from prospective and retrospective sampling will aid in understanding infections and diseases, including influenza, coronaviruses or other examples of known or unknown infectious agents, including new varieties, and body responses to disease and susceptibility to disease aspects. The collection of standard venipuncture samples places health care personnel at risk and is overly invasive and difficult for patients and individuals who have suffered pain or are under pressure and severe conditions and conditions.

The present invention may be embodied in other forms or carried out in other ways without departing from its spirit or essential characteristics. The present disclosure is, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims, and all changes which come within the meaning and range of equivalency are therefore intended to be embraced therein.

Various references are cited in this specification, each of which is incorporated herein by reference in its entirety.

Claims

1. A method for RNA profiling and analysis of a small volume sample from a patient or individual, comprising:

2. The method of claim 1, wherein RNA is isolated using an operation comprising:

3. The method of claim 2, wherein between steps (b) and (c), the RNA-containing resuspended pellet or the RNA-containing aqueous phase is contacted with a solution or column to remove residual sample cell debris and/or to homogenize the sample cell lysate.

4. The method of claim 1, wherein RNA is isolated using an operation comprising:

(f) Optionally further contacting the sample with a salt, a reducing agent, and/or a detergent;

(g) Contacting the sample with silica, silica-based solid phase or carboxylated magnetic beads capable of binding RNA and purifying RNA from other components in the sample, contacting the sample with the sample solution of (a) or (b); and

5. The method according to any one of claims 1-4, wherein the sample is a small volume blood sample.

6. The method according to any one of claims 1-5, wherein the small volume blood sample is collected by finger prick.

7. The method of any one of claims 1-6, wherein the sample volume is less than 500 μl, less than 300 μl, less than 250 μl, about 200-300 μl, less than 200 μl, about 100-300 μl, about 150-300 μl, about 100-250 μl, about 50-300 μl, less than 100 μl, less than 50 μl, less than 25 μl, 10 μl or less.

8. The method of any one of claims 1-7, wherein buffer and solution volumes are reduced to 20-40% or 20-30% or about 25% of the volume used to isolate RNA from a standard venipuncture blood sample.

9. The method of any one of claims 1-8, wherein the protease is proteinase K.

10. The method of any one of claims 1-9, further comprising sequencing RNA.

11. The method of claim 10, wherein a substantial amount of RNA species or RNA species of no interest are removed prior to sequencing.

12. The method of claim 11, wherein the globin mRNA, ribosomal RNA, or species-specific RNA is removed prior to sequencing.

13. The method of any one of claims 1-12, wherein the patient or individual has or is at risk of having a disease or infection.

14. The method according to any one of claims 1-13 for longitudinal screening by RNA profiling and analysis of small volume samples from one or more patients or individuals suffering from or at risk of or suspected of suffering from a disease or infection.

15. The method of claim 14, wherein the small volume samples are collected by finger prick in a continuous or regular manner or in specified increments of hours, days, weeks or months.

16. The method of claim 15, wherein the small volume blood sample is collected by finger prick in a continuous or regular manner or in specified increments of hours, days, weeks, or months.

17. A system or kit for RNA profiling and analysis of a small volume sample from a patient or individual, comprising:

(a) Means for self-collection of small volumes of sample by a patient or individual or non-medical personnel, including lancets, swabs or containers for washing, spitting or aspiration;

(c) One or more suitable labels for designating the name or identity of the patient or individual, the date of sample collection, and the time of sample collection.

18. The system or kit of claim 17, further comprising an envelope or mailing container for transporting the sample to a laboratory or facility for RNA isolation and analysis.

19. The system or kit of claim 17 for longitudinal RNA spectroscopy and analysis of a plurality of small volume samples continuously collected from a patient or individual over days, weeks or months, comprising:

(a) A multi-tool stack for self-collection of small volumes of samples by a patient or individual or by non-medical personnel, each comprising a lancet, swab, or container for washing, spitting, or aspiration;

20. The system or kit of any one of claims 17-19, wherein the volume of RNA stabilizing solution is less than 1ml, about 500 μl or less, about 300 μl or less, about 200-300 μl, about 250 μl, less than 200 μl, less than 100 μl, less than 50 μl, less than 25 μl, or 10 μl or less.

21. The system or kit of any one of claims 17-20, wherein the tube or container for receiving a small volume sample and containing the RNA stabilization solution has a total volume capacity of 1.5ml or less, 1.2ml or less, or 1ml or less.