CN109576350B

CN109576350B - Kit and method for simultaneously quantifying DNA and RNA and quality control method

Info

Publication number: CN109576350B
Application number: CN201910047187.3A
Authority: CN
Inventors: 陈力; 郑宗立; 高彦秋; 何云蔚; 陈淼; 赖家嘉
Original assignee: Helitec Ltd
Current assignee: Helitec Ltd
Priority date: 2019-01-18
Filing date: 2019-01-18
Publication date: 2021-01-29
Anticipated expiration: 2039-01-18
Also published as: CN109576350A

Abstract

The invention relates to a kit, a method and a quality control method for simultaneously quantifying DNA and RNA in a sample containing nucleic acid, belonging to the field of molecular biology. In the kit of the present invention, the nucleic acid comprises a housekeeping gene, the housekeeping gene comprises a first intron and two exons adjacent to the first intron, the two exons are a first exon and a second exon, respectively; the kit comprises an amplification primer capable of specifically annealing to a nucleic acid sequence of a first exon and a second exon, a first hybridization probe capable of hybridizing to the first intron, and a second hybridization probe capable of hybridizing to the first exon or the second exon, the first hybridization probe and the second hybridization probe carrying different labels. The invention also compares the quality control of the measured value of DNA and RNA which are quantified simultaneously with the high-throughput sequencing detection value, and provides a dual-quality control method for the quality of DNA and RNA.

Description

Kit and method for simultaneously quantifying DNA and RNA and quality control method

Technical Field

The invention relates to a kit, a method and a quality control method for simultaneously quantifying DNA and RNA, belonging to the field of molecular biology.

Background

Next Generation Sequencing (NGS) technology has revolutionized the field of genomics over the past decade. Each NGS run typically generates thousands of megabases of sequence information on hundreds of thousands to billions of DNA templates in parallel per sequencing run. The cost for human genome sequencing has currently reached a benchmark of $ 1000. The low cost and high throughput of NGS technology enables one to use nucleic acid sequencing as a clinical tool.

However, many challenges remain to achieve the desired cost, speed, analytical sensitivity and accuracy required for NGS clinical applications. Clinical samples, such as biopsy samples and Formalin Fixed Paraffin Embedded (FFPE) samples, provide only a small amount of starting material. NGS library construction can use DNA or RNA as an initial template, and is respectively suitable for detection of different gene mutation types. Nucleic acid in clinical samples is often stored at room temperature for a long time, and the nucleic acid, particularly RNA, in the nucleic acid is degraded in a large amount, so that the establishment of an RNA template library becomes difficult. The treatment process of formalin fixation of samples commonly used in clinic generates cross linking or destruction of nucleic acid, so that the efficiency of NGS library construction is reduced and the background of detection results is increased. Therefore, it is necessary to quickly and easily perform content and quality assessment of DNA and RNA templates before it takes much time and cost to construct NGS libraries.

Disclosure of Invention

The invention aims to provide a kit, a method and a kit for simultaneously quantifying DNA and RNA, and a quality control method of the DNA and the RNA.

In order to achieve the purpose, the invention adopts the technical scheme that:

in a first aspect, the present invention provides a kit for simultaneous quantification of DNA and RNA in a sample comprising nucleic acids, wherein said nucleic acids comprise a housekeeping gene comprising a first intron having a number of bases less than 300 and two exons adjacent to said first intron, the two exons being a first exon and a second exon, respectively; the kit comprises an amplification primer capable of specifically annealing to the nucleic acid sequences of the first and second exons, a first hybridization probe hybridizable to the first intron, and a second hybridization probe hybridizable to the first exon or the second exon, the first and second hybridization probes carrying different labels.

Preferably, the kit further comprises a PCR reaction solution and a reverse transcription reaction solution. Preferably, the PCR reaction solution comprises DNA polymerase, deoxynucleotide, inorganic salt and pH buffer solution; the reverse transcription reaction solution comprises reverse transcriptase, deoxynucleotide, inorganic salt and pH buffer solution.

Preferably, the kit further comprises a random primer or a poly-dT polynucleotide primer complementary to the polyA tail of the mRNA.

Preferably, the first hybridization probe and the second hybridization probe have luminescent groups with different luminescent wavelengths.

Preferably, the luminophore is FAM, ROX, HEX, CY5, TET or VIC.

Preferably, any of the following (a) to (e):

(a) the nucleic acid comprises genomic DNA or a fragment thereof;

(b) the nucleic acid comprises RNA;

(c) the nucleic acid comprises cDNA or a fragment thereof, preferably the cDNA is obtained by reverse transcription of total RNA, mRNA, miRNA or other non-coding RNA;

(d) the nucleic acid comprises genomic DNA and RNA;

(e) the nucleic acid comprises genomic DNA and cDNA.

Preferably, the sample is derived from a blood sample, a cell sample, a tissue sample, a food sample, an environmental sample or a biological sample.

Preferably, the kit further comprises instructions for use of the kit.

In a second aspect, the present invention provides a method for simultaneously quantifying DNA and RNA in a sample containing nucleic acid using the above-mentioned kit, comprising the steps of:

(1) selecting the housekeeping gene, and carrying out reverse transcription by taking the nucleic acid as a template to convert RNA in the nucleic acid into cDNA;

(2) taking a product after reverse transcription in the step (1) as a template, taking the amplification primer as a primer, and simultaneously adding the first hybridization probe and the second hybridization probe to perform PCR amplification to obtain an amplicon;

(3) detecting signals generated by the labels on the first hybridization probe and the second hybridization probe, and quantifying DNA and RNA in a sample containing nucleic acid by the method comprising: respectively recording Ct values corresponding to the markers on the first hybridization probe and the second hybridization probe, and analyzing the quality of the DNA and RNA in the sample through the absolute value and the difference value of the Ct values; wherein the Ct value is the corresponding amplification cycle number when the signal generated by the marker reaches a set detection threshold value.

In a third aspect, the present invention provides another method for simultaneously quantifying DNA and RNA in a sample containing nucleic acid using the above-described kit, comprising the steps of:

(3) taking the nucleic acid as a template, taking the amplification primer as a primer, and simultaneously adding the first hybridization probe and the second hybridization probe to perform PCR amplification;

(4) detecting signals generated by the labels on the first hybridization probe and the second hybridization probe in the steps (2) and (3) respectively, and quantifying DNA and RNA in the sample containing nucleic acid, wherein the quantification method comprises the following steps: recording Ct values corresponding to the markers on the first hybridization probe and the second hybridization probe in the step (2) and the step (3) respectively, analyzing the quality of DNA in the sample through the Ct values of the markers on the first hybridization probe and/or the second hybridization probe in the step (3), and analyzing the quality of RNA in the sample through the difference between the Ct value of the marker on the first hybridization probe in the step (2) and the Ct value of the marker on the first hybridization probe in the step (3); wherein the Ct value is the corresponding amplification cycle number when the signal generated by the marker reaches a set detection threshold value.

In a fourth aspect, the present invention provides a method for quality control of DNA and RNA in a sample containing nucleic acids, comprising the steps of:

selecting an reference gene from a nucleic acid, said reference gene comprising a first intron having a base number of less than 300, a second intron having a base number of greater than 300, two exons adjacent to said first intron, and two exons adjacent to said second intron; or selecting two reference genes from the nucleic acid, wherein one reference gene comprises a first intron with a base number of less than 300 and two exons adjacent to the first intron, and the other reference gene comprises a second intron with a base number of more than 300 and two exons adjacent to the second intron; wherein, two exons adjacent to the first intron are a first exon and a second exon respectively, and two exons adjacent to the second intron are a third exon and a fourth exon respectively;

simultaneously quantifying DNA and RNA in a sample containing nucleic acid by the method according to any one of claims 10 to 13 using an internal reference gene containing a first intron as a housekeeping gene;

a library establishing primer at the edge of the third exon is used for establishing a library by single-end anchoring of RNA with the nucleic acid as a template; measuring the DNA amplification product of the internal reference gene by using the base sequencing reading of the junction area of the second intron and the third exon, measuring the RNA amplification product of the internal reference gene by using the sequencing reading of the fourth exon, and judging the RNA library construction quality of the NGS library by using the ratio of the two sequencing readings;

and comprehensively analyzing the quality of the DNA and the RNA in the sample according to the result of the simultaneous quantification of the DNA and the RNA and the result of judging the RNA library building quality of the NGS library.

Compared with the prior art, the invention has the beneficial effects that: the invention provides a method and a kit for simultaneously quantifying DNA and RNA, and the kit and the method can be used for simultaneously analyzing the DNA and the RNA. In addition, the invention also compares the quality control of the measured value of DNA and RNA which are quantified simultaneously with the high-throughput sequencing detection value, and provides a double-quality control method for the quality of DNA and RNA.

Drawings

FIG. 1 is a schematic diagram of the method for controlling the quality of DNA and RNA in a sample containing nucleic acid according to the present invention;

FIG. 2 is a graph of the results of plotting the Ct difference between exon and intron probes of reverse transcription quantitative PCR and the ratio of RNA to DNA reading of reference gene in NGS sequencing in example 3 of the present invention;

FIG. 3 is a graph of the results of plotting the Ct difference between exon probes for reverse transcription quantitative PCR and non-reverse transcription quantitative PCR and the ratio of RNA to DNA read of reference gene in NGS sequencing in example 3 of the present invention;

FIG. 4 is a graph of the results of plotting the ratio of RNA to DNA concentration quantified using Qubit versus the ratio of RNA to DNA reads for a reference gene in NGS sequencing in example 3 of the present invention;

FIG. 5 is a graph of the results of plotting RNA concentration quantified using the Qubit versus the ratio of RNA to DNA reads for a reference gene in NGS sequencing in example 3 of the present invention.

Detailed Description

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, the molecular genetics, nucleic acid chemistry and molecular biology-related terms and laboratory procedures used herein are all terms and conventional procedures used extensively in the relevant art. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

The terms:

"nucleic acid" refers to a polymer of nucleotides of any length, and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or analogs thereof, or any substrate that can be incorporated into a polymer by a DNA or RNA polymerase.

"amplification" as used herein generally refers to the process of producing two or more copies of a desired sequence. Components of the amplification reaction may include, but are not limited to, for example, primers, polynucleotide templates, polymerases, nucleotides, dntps, and the like.

"polymerase chain reaction amplification" or "PCR amplification" refers to a method of amplifying a specific fragment or subsequence of a target double-stranded DNA in a geometric series. PCR is well known to those skilled in the art; see, for example, U.S. Pat. Nos. 4,683,195 and 4,683,202; and "PCR Protocols: A Guide to Methods" edited by Innis et al 1990. PCR amplification results in an exponential increase in the number of target nucleotide sequences.

"amplification product" or "amplicon" refers to an oligonucleotide produced by a PCR amplification reaction that is a copy of a portion of a particular target template nucleic acid strand and/or its complement, which corresponds in nucleotide sequence to the template nucleic acid sequence and/or its complement. The amplification product may also comprise a sequence specific for the primer and flanking the sequence of the target nucleic acid and/or its complement. Amplicons as described herein are typically double stranded DNA, although reference may be made to a single strand thereof.

"housekeeping gene", also known as housekeeping gene, refers to a class of genes that are stably expressed in all cells, the products of which are essential for maintaining the basic vital functions of the cell. The "reference gene" in the present invention corresponds to a "housekeeping gene". The expression of the reference gene and housekeeping gene is relatively abundant and stable in the source tissues of various samples, and the expression quantity is not regulated and controlled by biological conditions.

The term "exon" refers to the portion that is retained in the mature mRNA, i.e., the portion of the mature mRNA corresponding to a gene. Introns are the parts that are spliced out during mRNA processing and are not present in mature mRNA. Both exons and introns are for genes, the encoded part is an exon, the non-encoded part is an intron, and the intron has no genetic effect.

A "primer" is typically a short single-stranded polynucleotide, usually with a free 3' -OH group, that binds to a target of interest by hybridizing to the target sequence, and then facilitates polymerization of the polynucleotide complementary to the target.

"hybridization" and "annealing" are used interchangeably herein to refer to reactions that: wherein one or more polynucleotides react to form a complex that is stabilized by means of hydrogen bonds between bases of nucleotide groups. Hydrogen bonding can occur by Watson-Crick base pairing, Hoogstein binding, or by any other sequence specific means.

As used herein, the term "specifically anneals" refers to hybridization of a nucleic acid to a nucleic acid of complementary sequence. As used herein, a portion of a nucleic acid molecule can specifically hybridize to a complementary sequence on another nucleic acid molecule. That is, the entire length of a nucleic acid sequence need not necessarily hybridize to a portion of such sequence to "specifically hybridize" to another molecule, e.g., there may be a stretch of unhybridized nucleotides at the 5 'end of a molecule, while a stretch of the 3' end of the same molecule specifically hybridizes to another molecule.

A "hybridization probe" is a small piece of single-stranded DNA (a dozen to several hundred bases) that is used to detect a nucleic acid sequence complementary thereto. The hybridization probe carries a signal molecule, such as an isotope or a fluorescently labeled nucleic acid molecule, which can be detected. When the probe is hybridized with a sample, the probe and a nucleic acid (DNA or RNA) sequence complementary thereto are closely linked by hydrogen bonds.

In the present invention, "first", "second", and the like in "first hybridization probe", "second hybridization probe", "first exon", "second exon", "third exon", "fourth exon", "first intron", "second intron", and the like are only for the purpose of distinguishing hybridization probes, exons, introns, and the like, and are not limited in number.

"reverse transcription" is the process of synthesizing DNA by reverse transcriptase using RNA as a template. Is a special mode of DNA biosynthesis. Reverse transcription is a process of extracting DNA genetic information by taking RNA as a template in the process of genetic engineering.

"random primer" means that when it is difficult to copy a full-length mRNA due to the fact that a specific mRNA has a sequence for terminating a reverse transcriptase, a nonspecific primer of random hexamer can be used to copy the full-length mRNA.

A "reaction system" is a combination of components (e.g., one or more polypeptides, nucleic acids, and/or primers) that performs a particular reaction, such as a primer extension reaction or a PCR amplification reaction, under suitable conditions.

"library" refers to a collection of nucleic acid sequences.

The terms "determining," "detecting," "measuring," "evaluating," "assessing," "validating," and "analyzing" are used interchangeably herein to refer to any form of measurement, and include determining the presence or absence of an element. These terms include quantitative determinations and/or qualitative determinations.

It is to be understood that the aspects and embodiments of the invention described herein include aspects and embodiments "consisting of …" and/or "consisting essentially of …".

As used herein, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

As understood by those skilled in the art, reference herein to a value or parameter of "about" includes (and describes) embodiments that relate to that value or parameter per se. For example, a description of "about X" includes a description of "X".

Where a range of values is provided, it is understood that each intervening value, to the extent that there is no such intervening value, to the extent that there is provided a range of values between the upper and lower limits of that range, and any other stated or intervening value in that range, is encompassed within the scope of the disclosure. Where a stated range includes an upper limit or a lower limit, ranges excluding either of those included limits are also included in the disclosure.

Unless otherwise indicated, the invention is carried out using standard procedures as described in, for example, "Molecular Cloning: A Laboratory Manual (3 rd edition)" by Sambrook et al (New York Cold spring harbor Press, U.S.A., 2001); and "Basic Methods in Molecular Biology" by Davis et al (Elsevier Science, N.Y., 1995).

In order to examine the quality of DNA and RNA in a sample containing nucleic acid so as to improve the efficiency of subsequent high-throughput sequencing, in one embodiment of the invention, the invention provides a kit for simultaneously quantifying the DNA and RNA in the sample containing nucleic acid, wherein the nucleic acid comprises a housekeeping gene, the housekeeping gene comprises a first intron with the base number less than 300 and two exons adjacent to the first intron, and the two exons are a first exon and a second exon respectively; the kit comprises an amplification primer capable of specifically annealing to the nucleic acid sequences of the first and second exons, a first hybridization probe hybridizable to the first intron, and a second hybridization probe hybridizable to the first exon or the second exon, the first and second hybridization probes carrying different labels.

In the kit of the present invention, the first intron is short in length, and the short length means that the length of the amplification unit formed by the first intron and the adjacent two exons is suitable for efficient amplification. The number of bases of the first intron is required to be less than 300, and if the number of bases is more than 300, PCR amplification imbalance may be caused, for example, the number of bases of the first intron may be 75. The amplification primers capable of specifically annealing to the nucleic acid sequences of the first exon and the second exon are respectively positioned on different exons of one gene, the gDNA and the cDNA template can be amplified, and the size of the fragment of the generated amplicon is in the efficient amplification range of quantitative PCR reaction. The first hybridization probe and the second hybridization probe are both located in the amplicon range defined by the forward and reverse amplification primers. In the reverse transcription quantitative PCR process, the first hybridization probe acts on the intron sequence of the gene and can detect only gDNA signal, and the second hybridization probe acts on the exon sequence of the gene and can detect the sum of gDNA and cDNA signals (described as RT _ VIC).

By using a pair of amplification primers and two specific probes in the kit, the gDNA signal and the sum of the gDNA and cDNA signals can be simultaneously determined by reverse transcription quantitative PCR so as to realize the simultaneous quantification of the DNA and the cDNA.

As a preferred mode of one embodiment of the kit of the present invention, the kit of the present invention further comprises a PCR reaction solution and a reverse transcription reaction solution. More preferably, the PCR reaction solution comprises DNA polymerase, deoxynucleotide, inorganic salt and pH buffer solution; the reverse transcription reaction solution comprises reverse transcriptase, deoxynucleotide, inorganic salt and pH buffer solution. Further, the kit may further comprise a random primer or a poly-dT polynucleotide primer complementary to the polyA tail of the mRNA.

One of the above reverse transcriptases, RNA-dependent, is a template DNA polymerase. In the reverse transcription process, the adopted reaction reagents are the reverse transcription reaction liquid and oligonucleotide primers. The oligonucleotide primer may be a gene specific primer, such as one of the amplification primers capable of specifically annealing to the nucleic acid sequences of the first and second exons, or may be either a random primer or a poly-dT polynucleotide primer complementary to the polyA tail of the mRNA.

The pH buffer solution in the PCR reaction solution is used for adjusting the pH value of a PCR reaction system, and the pH buffer solution in the reverse transcription reaction solution is used for adjusting the pH value of a reverse transcription system. One skilled in the art can select suitable pH buffer and inorganic salt according to routine knowledge, for example, pH buffer is selected from buffer solution containing Tris (hydroxymethyl) aminomethane (Tris), such as Tris-HCl, etc.; the inorganic salt is selected from magnesium chloride, etc. All components of the PCR reaction system and the reverse transcription system can be purchased or synthesized from a biological reagent manufacturer.

In the kit of the present invention, the label on the hybridization probe is a detectable signal molecule, which may be a fluorescent label, or other labels, such as radioactive isotope (usually phosphorus-32) and the like. In order to facilitate the fluorescent quantitative analysis, the marker selected by the invention is a luminescent group, namely a fluorescent marker. In order to make the two probes generate different signals respectively, so that the two signals can be simultaneously detected by a fluorescent quantitative PCR instrument capable of detecting a plurality of wavelengths, the first hybridization probe and the second hybridization probe are provided with luminescent groups with different luminescent wavelengths. Preferably, the luminophore is FAM, ROX, HEX, CY5, TET or VIC. More preferably, the label carried on the first hybridization probe is FAM and the label carried on the second hybridization probe is VIC. FAM and VIC probes can be ordered and synthesized from a bioreagent manufacturer, such as Thermo Fisher.

Preferably, any of the following (a) to (e):

(a) the nucleic acid comprises genomic DNA or a fragment thereof;

(b) the nucleic acid comprises RNA;

(d) the nucleic acid comprises genomic DNA and RNA;

(e) the nucleic acid comprises genomic DNA and cDNA.

The kit of the present invention can be used for various nucleic acids in a sample. In some embodiments, the nucleic acid comprises RNA. In some embodiments, the nucleic acid comprises a mixture of genomic DNA and RNA.

In some embodiments, the nucleic acid template is derived (e.g., fragmented) from a nucleic acid, such as full-length chromosomal DNA or full-length mRNA, that is greater in length than the optimal read length for the NGS method or platform.

In some embodiments, the nucleic acid comprises cDNA or a fragment thereof. In some embodiments, the cDNA is obtained by reverse transcription of total RNA or portions thereof (such as mRNA, miRNA, or other non-coding RNA). In some embodiments, the cDNA is single stranded, e.g., at least any one or more of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the cDNA is single stranded. In some embodiments, the nucleic acid comprises a double-stranded cDNA.

In some embodiments, the nucleic acid comprises gDNA or a fragment thereof. In some embodiments, the gDNA is single stranded, e.g., at least about any one or more of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the gDNA is single stranded. In some embodiments, the nucleic acid comprises double-stranded gDNA.

In some embodiments, the nucleic acid comprises a mixture of cDNA and gDNA. In some embodiments, the weight ratio between cDNA and gDNA is greater than about any one of 1:10, 1:5, 1:3, 1:2, 1:1, 2:1, 3:1, 5:1, 10:1, or more.

In some embodiments, the nucleic acid comprises no more than about any one of 1000ng, 500ng, 200ng, 100ng, 50ng, 40ng, 30ng, 25ng, 20ng, 15ng, 10ng, 5ng, 4ng, 3ng, 2ng, 1ng or less of a nucleic acid template (such as cDNA, gDNA, RNA, combinations thereof, or total nucleic acid).

In some embodiments, a sample of the invention is derived from a cell or tissue sample. In some embodiments, a sample of the invention is derived from a cell line sample or from cultured cells. In some embodiments, a sample of the invention is derived from a genetically engineered cell line. In some embodiments, a sample of the invention is derived from a tumor cell.

In some embodiments, the sample of the invention is obtained from a food sample, an environmental sample, or a biological sample. In some embodiments, a sample of the invention is derived from a biological sample from an individual. In some embodiments, a sample of the invention is derived from a biological sample in need of treatment for a disease (such as cancer). In some embodiments, a sample of the invention is a diagnostic sample obtained from an individual. In some embodiments, the sample of the invention is derived from a biological sample from a healthy individual. In some embodiments, a sample of the invention is derived from a genetically engineered animal (such as a mouse, rat, or non-human primate).

In some embodiments, the biological sample further comprises proteins, cells, fluids, biological fluids, preservatives, and/or other substances. As non-limiting examples, the sample may be a cheek swab, blood, serum, plasma, sputum, cerebrospinal fluid, urine, tears, alveolar isolate, pleural fluid, pericardial fluid, cyst fluid, tumor tissue, biopsy, saliva, aspirate, or a combination thereof. In some embodiments, the biological sample is obtained by resection or biopsy.

In some embodiments, a sample of the invention is derived from a blood sample of an individual. In some embodiments, a sample of the invention is derived from a peripheral blood mononuclear cell (PMBC) sample of an individual. In some embodiments, a sample of the invention is derived from a portion of immune cells (such as T cells, NK cells, or B cells) in a blood sample of an individual. In some embodiments, the nucleic acid template is cell-free DNA. In some embodiments, the nucleic acid template is cell-free DNA derived from a blood sample of the individual. In some embodiments, the nucleic acid template is circulating tumor DNA (i.e., ctDNA). In some embodiments, the nucleic acid template is derived from circulating tumor cells of a blood sample of the individual.

In some embodiments, a sample of the invention is derived from a biopsy sample of an individual. In some embodiments, a sample of the invention is derived from a tumor biopsy, such as an untreated biopsy or a treated biopsy. In some embodiments, the sample of the invention is derived from formalin-fixed and/or paraffin-embedded biopsy tissue of an individual.

In some embodiments, the biological sample is obtained from an individual in need of treatment for a disease associated with a genetic alteration (such as cancer or a genetic disease). In some embodiments, the target sequence is known to be present in a gene associated with a disease. In some embodiments, the biological sample is obtained from an individual in need of treatment for cancer. In some embodiments, the biological sample comprises tumor cells at one or more tumor sites in the individual.

In some embodiments, the biological sample is freshly collected from the individual. In some embodiments, the biological sample is stored for a period of time, such as at least about any one of 1 day, 1 week, 1 month, 3 months, 6 months, 1 year or more, prior to use in the methods described herein. In some embodiments, the biological sample is a Formalin Fixed Paraffin Embedded (FFPE) sample. In some embodiments, the biological sample is used directly as a nucleic acid sample in the methods described herein. In some embodiments, the biological sample is pre-treated by dilution and/or suspension in a solution. In some embodiments, the biological sample is obtained from a subject and stored or processed prior to use in the methods described herein. For example, biological samples may be embedded in paraffin, refrigerated, or frozen. The frozen biological sample may be thawed prior to use. Other exemplary treatments or processes of biological samples include, but are not limited to, centrifugation, filtration, sonication, homogenization, heating, freeze-thawing, contact with a preservative (e.g., an anticoagulant or nuclease inhibitor), and any combination thereof. In some embodiments, the biological sample is treated with a chemical and/or biological reagent. Chemical and/or biological reagents can be used to protect and/or maintain the stability of a biological sample or nucleic acid template contained therein during processing and/or storage. In some embodiments, chemical and/or biological reagents may be used to release nucleic acid templates from other components of a biological sample. As a non-limiting example, a blood sample may be treated with an anticoagulant prior to use in obtaining a nucleic acid sample for use in the methods described herein. Methods and procedures for processing, preserving or handling biological samples, as well as methods of isolating nucleic acids from biological or cellular samples for nucleic acid analysis, are well known to the skilled person.

In some embodiments, the nucleic acid templates in a biological sample or nucleic acid sample may be isolated, enriched, or purified prior to use in the methods described herein. Suitable methods for isolating, enriching or purifying nucleic acids from a sample may be used.

To facilitate use of the kit of the invention, the kit further comprises instructions for use of the kit.

The kits of the invention may also comprise one or more additional components such as containers, cofactors, or additional reagents such as denaturants. The kit components may be packaged together.

In one embodiment of the present invention, the present invention provides a method for simultaneously quantifying DNA and RNA in a sample containing nucleic acid using the above-described kit, comprising the steps of:

In step (1) of the above method of the present invention, a mixed nucleic acid containing genomic DNA (gDNA) and RNA is reverse transcribed, the reverse transcription reaction is initiated by an oligonucleotide bound to the RNA template, and DNA with a complementary sequence is replicated by catalysis of the reverse transcriptase using deoxynucleotides as a reaction substrate, i.e., RNA therein is converted into complementary DNA (cDNA), and the resulting reverse transcription product is a mixture of genomic DNA and cDNA. gDNA differs from cDNA in that gDNA contains exon and intron sequences, whereas cDNA contains only exon sequences. Wherein, the reverse transcription can be performed under the following conditions: the components of the reverse transcription system are mixed with the nucleic acid to be quantified, and incubated at a certain temperature (e.g., 42 to 55 ℃) for a certain period of time (e.g., 5 minutes to two hours) to obtain complementary DNA (cDNA) of the RNA template. Subsequently, a quantitative amplification reaction (e.g., qPCR) is performed using a pair of amplification primers and two differently labeled probes (e.g., FAM probe and VIC probe). In the process of quantitative amplification reaction, a pair of positive and negative amplification primers are respectively positioned on different exons of a gene, the reaction is initiated by the amplification primers combined with a DNA template (comprising gDNA and cDNA templates), and a new DNA chain is copied by using deoxynucleotide as a reaction substrate under the catalysis of DNA polymerase enzyme. The new DNA chain becomes a template of the next round of reaction, so that the reaction product amplifies the specific segment of the target double-stranded DNA in a geometric series mode, and the segment size of the generated amplicon is in the efficient amplification range of the quantitative PCR reaction. Wherein the first hybridization probe acts on an intron to detect only gDNA signals, and the second hybridization probe acts on an exon to detect the sum of gDNA and cDNA signals (RT _ VIC).

In some embodiments, reagents for a quantitative PCR system (i.e., PCR reaction solution, amplification primers, first hybridization probe, and second hybridization probe) may be added after reverse transcription is complete. In other embodiments, quantitative PCR system components (i.e., PCR reaction, amplification primers, first hybridization probe, and second hybridization probe) may also be pre-mixed in the reverse transcription system, referred to as "one-step reverse transcription PCR," which is well known to those skilled in the art. The "one-step reverse transcription PCR" system is first set at temperature and time suitable for reverse transcription, such as 42-55 deg.c for 5 min-two hr, to obtain complementary DNA (cDNA) of RNA template. Under this condition, the thermostable DNA polymerase used for quantitative PCR does not function. The system is then heated to a PCR cycle at which the thermal melting temperature (e.g., 80 to 100℃) deactivates the reverse transcriptase and terminates the reverse transcription reaction.

In the ideal case of quantitative PCR, the Ct value is inversely proportional to the base 2 logarithm of the DNA content, i.e.the Ct value decreases by 1 for every doubling of the DNA content. In practical application, the Ct value is affected by the binding efficiency of the probe to the template, the light intensity of different fluorophores, the amplification efficiency, incidental factors and the like. Generally, the relative amounts of two nucleic acids can still be determined by comparing the Ct values. The term "Ct value low" is used herein to equate to "signal strong" and vice versa.

In the method of the present invention for simultaneous quantification of DNA and RNA in a sample containing nucleic acids, the signal from the first hybridization probe is evaluated gDN in the sample, and the difference between the signal from the second hybridization probe and the signal from the first hybridization probe is evaluated RNA in the sample. For example, if a FAM group is used to label a first hybridization probe and a VIC group is used to label a second hybridization probe, two Ct values, designated RT _ FAM and RT _ VIC, are obtained by reverse transcription quantitative PCR. RT _ FAM reflects the signal generated by the DNA template, and the difference between RT _ VIC and RT _ FAM reflects the signal generated by RNA. The absolute value of the signal and the threshold value of the difference can be judged by data accumulation and experience setting:

in some samples, the RT _ FAM signal is strong, and the RT _ VIC value is obviously lower than that of RT _ FAM, which indicates that the quality of DNA and RNA is good;

in some samples, the RT _ FAM signal is strong, and the difference between RT _ VIC and RT _ FAM is not obvious, which indicates that the DNA quality is good and the RNA quality is poor;

in some samples, the RT _ FAM signal was weak and the difference between RT _ VIC and RT _ FAM was not significant, indicating poor DNA and RNA quality;

in some samples, the RT _ FAM signal is weak, and the RT _ VIC value is obviously lower than that of RT _ FAM, which indicates that the RNA quality is good; for nucleic acid samples that have not been subjected to active intervention (e.g., degradation treatment with DNA nucleases, or extraction of only RNA), since RNA is less stable than DNA, good RNA can be used to infer that the DNA in the sample is good, whereas the weak RT FAM signal is due to strong competitive inhibition of RNA.

In one embodiment of the present invention, the present invention provides another method for simultaneously quantifying DNA and RNA in a sample containing nucleic acid using the above-described kit, comprising the steps of:

The step (3) is a quantitative PCR reaction system without reverse transcription. In this step, the reagents for quantitative PCR system (i.e., PCR reaction solution, amplification primer, first hybridization probe and second hybridization probe) may be added alone or in combination with some or all of the reagents required for reverse transcription. When reagents required for reverse transcription are added simultaneously, this can be achieved by omitting reverse transcriptase or other necessary components or omitting the reverse transcription incubation step and heating directly into the PCR cycle so that the RNA is not reverse transcribed. All signal values obtained by the quantitative PCR without reverse transcription in the step (3) are from gDNA, and can be used for evaluating the gDNA in a sample; the difference between the sum of gDNA and cDNA signals of the reverse transcription quantitative PCR of steps (1) and (2) and the gDNA value of the same probe of the reaction tube without reverse transcription of step (3) allows the RNA in the sample to be evaluated.

In another preferred method of the present invention for simultaneously quantifying DNA and RNA in a sample containing nucleic acid using the above-described kit, if FAM groups are used to label the first hybridization probe and VIC groups are used to label the second hybridization probe, two Ct values, designated RT _ FAM and RT _ VIC, are obtained by reverse transcription quantitative PCR in step (1) and step (2), and two additional Ct values, designated NR _ FAM and NR _ VIC, are obtained by quantitative PCR without reverse transcription in step (3):

the DNA in the sample can be measured with NR _ FAM and/or NR _ VIC, with lower Ct values indicating good DNA quality. This signal is not interfered by inhibition by RNA.

The difference between RT _ VIC and NR _ VIC can be used to judge the RNA in the sample, and if the RT _ VIC value is obviously lower than NR _ VIC, the RNA quality is good. The RNA quality can be judged by the difference between RT _ FAM and NR _ FAM. Better RNA will have RT _ VIC that inhibits RT _ FAM, resulting in higher readings than NR _ FAM.

In the amplification reaction, the reverse-transcribed RNA forms competitive inhibition on DNA signals, so the increase of RT _ VIC signals is accompanied with the decrease of RT _ FAM, and the quality of RNA in the sample can be evaluated by the relative decrease of RT _ FAM and NR _ FAM. The simultaneous use of reverse transcription quantitative PCR and reverse transcription-free quantitative PCR may yield more data points, but may require more reagent cost and operating cost, which may be unnecessary in some cases.

In the above method for controlling the quality of DNA and RNA in a sample containing nucleic acid, the first intron is a shorter intron, and the second intron is a longer intron. The first intron is a shorter intron, which means that the length of the amplification unit formed by the portions of the adjacent two exons is suitable for efficient amplification, the number of bases of the first intron is required to be less than 300, and if the number of bases of the first intron is more than 300, PCR amplification can be unbalanced, for example, the number of bases of the first intron can be 75. The second intron is a longer intron, which means that the length of the second intron exceeds the average fragmentation length of the nucleic acid sequenced in two generations, such as more than 300 bases, specifically 1608 bases, and if the length of the second intron is shorter than 300 bases, the second intron may not be capable of distinguishing large-fragment DNA reads from RNA reads. The first intron and the second intron may be adjacent or non-adjacent if they are located on the same gene.

In the method for controlling the quality of the DNA and the RNA in the sample containing the nucleic acid, quantitative PCR and NGS sequencing reading become associated leading prediction points and subsequent analysis points respectively, and the quality control mode of constructing the RNA library is formed together. The method for controlling the quality can be used for controlling and evaluating the quality of nucleic acid before carrying out gene detection (such as gene sequencing or various PCR) on a sample. Such genetic testing generally involves the replication of nucleic acid templates, with requirements for nucleic acid content and quality, whereas nucleic acid quantification methods using spectrometers can only determine nucleic acid content but cannot assess nucleic acid quality. The quality control method needs to perform replication amplification on the nucleic acid, and if the content of the nucleic acid is high and the integrity is good, the amplification effect is good. Applications such as in Next Generation Sequencing (NGS) pooling are:

a) the amplicon has a fragment size range suitable for high throughput sequencing and thus can be used for DNA and/or RNA quality control of starting nucleic acids constructed from NGS libraries.

b) If the DNA signal of the sample is good, which indicates that the nucleic acid is complete, it is suitable to construct the NGS library by using the DNA as a template.

c) If the extracted RNA content is high and the extracted RNA is complete when the sample is fresh, the method is suitable for NGS library construction with RNA as a template; otherwise, the RNA content is low or the RNA is degraded, and the library building is not suitable.

It should be noted that the present invention has a strong correlation between the result of simultaneously quantifying DNA and RNA in a sample containing nucleic acid and the quality of the detection result of genes such as NGS, but does not indicate that the two are completely matched. The method for controlling the quality of the nucleic acid can still obtain a good detection result when the nucleic acid with low quality is judged, and vice versa, so that the method for controlling the quality of the nucleic acid is suitable for quickly and conveniently evaluating whether the quality of a sample is suitable for high-throughput sequencing gene detection with high requirements on operation and cost.

The method for controlling the quality of DNA and RNA in a sample containing nucleic acid according to the present invention is shown in FIG. 1.

Example 1

This example describes the kit of the present invention for simultaneous quantification of DNA and RNA in a sample containing nucleic acids. Wherein, the selected housekeeping gene is human CHMP2A gene, the shorter intron in the CHMP2A gene is the No. 3 intron (the base number is 75), and the longer intron is the No. 2 intron (the base number is 1608). The composition of the kit of the present invention is shown in table 1, and the sequences of the amplification primers and the probes in the kit of the present invention are shown in table 2. Wherein, the amplification primer 1 and the amplification primer 2 can anneal to the nucleic acid sequences of two exons adjacent to the intron 3 in the CHMP2A gene.

Table 1 composition of the kit.

TABLE 2 sequences of amplification primers and probes

Example 2

This example describes the simultaneous quantification of DNA and RNA in a sample containing nucleic acids using the kit described in example 1, i.e., the method of using the kit described in example 1. The method adopts a fluorescent quantitative PCR instrument as equipment, and comprises the following specific steps:

pretreatment of reagents before first use: 2 tubes of 50 XPrimer & Probe Mix were added to 2 XPPreQC DNA and 2 XPPreQC RNA respectively and mixed, labelled 2 XPPreQC DNA Mix and 2 XPPreQC RNAmix.

1) The reagents were formulated and mixed according to tables 3 and 4 below:

TABLE 3 quantitative PCR System without reverse transcription

Components	Volume of
		2×PreQC DNA Mix	5.0μL
Nuclease-free deionized water	4μL
		Nucleic acid sample to be tested	1μL
Total volume	10.0μL

TABLE 4 reverse transcription quantitative PCR System

Components	Volume of
		2×PreQC RNA Mix	5.0μL
Nuclease-free deionized water	4μL
		Nucleic acid sample to be tested	1μL
Total volume	10.0μL

2) Instantaneous centrifugation, computer program as shown in Table 5 below

TABLE 5

3) Setting parameters: taking the QuantStudio 3 instrument as an example, the threshold of FAM is set to 20000 and the threshold of VIC is set to 10000.

Example 3

This example describes the quality assessment process of nucleic acids from a batch of clinical tumor tissue samples: the samples were total nucleic acids extracted from 244 tumor tissues, and quantitative PCR values were measured using the kit described in example 1, and DNA and RNA were quantified using a Qubit manufactured by Thermo Fisher, respectively. The nucleic acid is used for targeting NGS library construction by using RNA and DNA as templates through a single-end anchoring method, and the detection range of the nucleic acid comprises a gene which is abundant in each type of tissue and stable in expression as an internal reference. The amplicon of the gene starts from the border region of one exon, the sequencing reading of the adjacent intron is used for measuring the signal from the DNA template, the sequencing reading of the exon positioned at the other end of the intron is used for measuring the signal from the RNA template, and the ratio of the two signals is used for measuring the RNA detection quality of the NGS library.

The difference in Ct between exon and intron probes (x-axis, RT _ VIC-RT _ FAM) of reverse transcription quantitative PCR was plotted against the ratio of RNA to DNA read of reference gene in NGS sequencing (y-axis, NGS: RNA2DNA), and the results are shown in FIG. 2. Data were collected from NGS libraries of 244 clinical samples and samples with misleading data were discarded. In FIG. 2, the RNA detection quality is considered to be good when the RNA to DNA reading ratio of the NGS reference gene is 0.3 or more (right side of the vertical solid line). As can be seen from fig. 2, when the difference between RT _ VIC and RT _ FAM is less than-0.5 (below the horizontal dotted line), most of the samples fall on the right side of the vertical solid line, and all the sample points (circled by the oval dotted line in the figure) of the NGS completely free from RNA reading are located above or on the boundary of the horizontal dotted line, and are determined as nucleic acid-disqualified, which indicates that they can be used as the pilot quality control indicator of NGS detection quality.

The difference in Ct values for exon probes for reverse transcription quantitative PCR and non-reverse transcription quantitative PCR (x-axis, RT _ VIC-NR _ VIC) was plotted against the ratio of RNA to DNA read for reference gene for NGS sequencing (y-axis, NGS: RNA2DNA), and the results are shown in FIG. 3. Data were collected from NGS libraries of 244 clinical samples and samples with misleading data were discarded. The RNA detection quality is considered to be better when the RNA to DNA reading ratio of the NGS reference gene is more than 0.3 (right side of the vertical solid line). As can be seen from fig. 3, when the difference between RT _ VIC and NR _ VIC is less than-0.5 (below the horizontal dotted line), most of the samples fall on the right side of the vertical solid line, and all of the sample points (circled by the oval dotted line in the figure) where no RNA reading is found in the NGS are located above or on the boundary of the horizontal dotted line, and are determined as nucleic acid-disqualified, which indicates that they can be used as the pilot quality control indicator for NGS detection quality.

The ratio of RNA to DNA concentration quantified using a Qubit (x-axis, Qubit: RNA/DNA) is plotted against the ratio of reference gene RNA to DNA read in NGS sequencing (y-axis, NGS: RNA2DNA), and the results are shown in FIG. 4. Data were collected from NGS libraries of 244 clinical samples and samples with misleading data were discarded. The RNA detection quality is considered to be better when the RNA to DNA reading ratio of the NGS reference gene is more than 0.3 (right side of the vertical solid line). As can be seen from fig. 4, a horizontal line cannot be drawn in the figure, so that most of the samples above the horizontal line fall on the right side of the vertical solid line relative to most of the samples below the vertical solid line, and the sample points (circled by the oval dashed lines in the figure) of the NGS reference gene without RNA reading are scattered in the y-axis direction, so that whether the nucleic acid is qualified or not cannot be judged, which indicates that the ratio of RNA to DNA concentration is merely quantified without considering that the nucleic acid quality cannot be used as the pilot quality control index of the NGS detection quality.

The RNA concentration quantified using the Qubit (x-axis, Qubit: RNA (ng/ul)) was plotted against the reference gene RNA to DNA read ratio (y-axis, NGS: RNA2DNA) in NGS sequencing, with the results shown in FIG. 5. Data were collected from NGS libraries of 244 clinical samples and samples with misleading data were discarded. The RNA detection quality is considered to be better when the RNA to DNA reading ratio of the NGS reference gene is more than 0.3 (right side of the vertical solid line). As can be seen from fig. 5, it can be seen that a horizontal line cannot be made so that most of the samples below the horizontal line fall on the right side of the vertical solid line, and the sample points (circled by the oval dashed lines in the figure) of the NGS reference genes without RNA reading are scattered in the y-axis direction to fail to determine whether the nucleic acids are qualified, which indicates that quantification is performed only by RNA concentration without considering the quality of the nucleic acids as a pilot quality control indicator of the NGS RNA detection quality.

Three samples were taken and analyzed, and the results are shown in table 6. As can be seen from table 6, the Qubit quantification was normal for all three samples: the ratio of the Qubit to the RNA/DNA is more than 1, the ratio of the Qubit to the DNA is more than 10ng/ul, and the ratio of the Qubit to the RNA is more than 10 ng/ul. However, the former two libraries L18-00149T and L18-00152T have good NGS RNA detection effect (NGS: RNA/DNA >1), and the third library L18-00194T has poor NGS RNA detection effect (NGS: RNA/DNA ═ 0.14), which indicates that NGS and Qubit are not quantitatively matched, and the measurement result of the method is matched: the RT _ VIC-RT _ FAM and RT _ VIC-NR _ VIC values of the first two libraries were below the set threshold (-0.5), and the RT _ VIC-RT _ FAM and RT _ VIC-NR _ VIC values of the third library were above-0.5.

TABLE 6

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

SEQUENCE LISTING

<110> Shenzhen Hengte Gene Co., Ltd

<120> kit, method and quality control method for simultaneous quantification of DNA and RNA

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<170> PatentIn version 3.3

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Claims

1. A method for controlling the quality of DNA and RNA in a sample containing nucleic acid, which is characterized by comprising the following steps:

s1, selecting an internal reference gene from nucleic acid, wherein the internal reference gene comprises a first intron with the base number of less than 300, a second intron with the base number of more than 300, two exons adjacent to the first intron and two exons adjacent to the second intron; wherein, two exons adjacent to the first intron are a first exon and a second exon respectively, and two exons adjacent to the second intron are a third exon and a fourth exon respectively;

s2, simultaneously quantifying DNA and RNA in a sample containing nucleic acid by taking an internal reference gene containing a first intron as a housekeeping gene;

s3, a library establishing primer at the edge of a third exon is used for establishing a library by RNA single-end anchoring with the nucleic acid as a template; measuring the DNA amplification product of the internal reference gene by using the base sequencing reading of the junction area of the second intron and the third exon, measuring the RNA amplification product of the internal reference gene by using the sequencing reading of the fourth exon, and judging the RNA library construction quality of the next generation of sequencing library by using the ratio of the two sequencing readings;

s4, comprehensively analyzing the quality of the DNA and the RNA in the sample according to the result of the simultaneous quantification of the DNA and the RNA and the result of judging the RNA library building quality of the NGS library;

wherein the step S2 of simultaneously quantifying DNA and RNA in the sample containing nucleic acid specifically comprises the following steps:

(2) taking a product obtained after reverse transcription in the step (1) as a template, taking an amplification primer as a primer, and simultaneously adding a first hybridization probe and a second hybridization probe to perform PCR amplification to obtain an amplicon;

(3) detecting signals generated by the labels on the first hybridization probe and the second hybridization probe, and quantifying DNA and RNA in a sample containing nucleic acid by the method comprising: respectively recording Ct values corresponding to the markers on the first hybridization probe and the second hybridization probe, and analyzing the quality of the DNA and RNA in the sample through the absolute value and the difference value of the Ct values; wherein the Ct value is the corresponding amplification cycle number when the signal generated by the marker reaches a set detection threshold value;

the amplification primers are capable of specifically annealing to the nucleic acid sequences of the first and second exons; the first hybridization probe is hybridizable to a first intron; the second hybridization probe can be hybridized with the first exon or the second exon, and the first hybridization probe and the second hybridization probe are provided with different labels;

the housekeeping gene is human CHMP2A gene.

2. A method for controlling the quality of DNA and RNA in a sample containing nucleic acid, which is characterized by comprising the following steps:

(3) taking the nucleic acid as a template, taking an amplification primer as a primer, and simultaneously adding a first hybridization probe and a second hybridization probe to perform PCR amplification;

(4) detecting signals generated by the labels on the first hybridization probe and the second hybridization probe in the steps (2) and (3) respectively, and quantifying DNA and RNA in the sample containing nucleic acid, wherein the quantification method comprises the following steps: recording Ct values corresponding to the markers on the first hybridization probe and the second hybridization probe in the step (2) and the step (3) respectively, analyzing the quality of DNA in the sample through the Ct values of the markers on the first hybridization probe and/or the second hybridization probe in the step (3), and analyzing the quality of RNA in the sample through the difference between the Ct value of the marker on the first hybridization probe in the step (2) and the Ct value of the marker on the first hybridization probe in the step (3); wherein the Ct value is the corresponding amplification cycle number when the signal generated by the marker reaches a set detection threshold value;

the housekeeping gene is human CHMP2A gene.