WO2016161023A1 - Procédés d'isolement d'arn de grande qualité à partir d'échantillons fixés - Google Patents

Procédés d'isolement d'arn de grande qualité à partir d'échantillons fixés Download PDF

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WO2016161023A1
WO2016161023A1 PCT/US2016/025077 US2016025077W WO2016161023A1 WO 2016161023 A1 WO2016161023 A1 WO 2016161023A1 US 2016025077 W US2016025077 W US 2016025077W WO 2016161023 A1 WO2016161023 A1 WO 2016161023A1
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sample
cells
rna
lysis buffer
fixed
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PCT/US2016/025077
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Sumin JANG
Elliot R. THOMSEN
Adele M. DOYLE
Boaz P. LEVI
Sharad Ramanathan
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President And Fellows Of Harvard College
Allen Institute
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis

Definitions

  • RNA expression in cells and tissues is needed for many clinical and research studies.
  • Formaldehyde fixation of cells is a common method to preserve the intracellular nucleic acid and protein content prior to analysis.
  • recovering RNA from cells that have been formaldehyde fixed results in degraded and chemically modified fragments of RNA, rather than a distribution of full length RNA typical of un-fixed cells.
  • These degraded fragments of RNA can be biased (i.e., unrepresentative of true abundance levels in the cell), difficult to identify (when fragments are short or map to multiple regions), and difficult to quantify (due to incomplete or biased recovery and lack of mapping confidence). Accordingly, improved methods for recovering RNA from fixed cells and tissues are needed.
  • RNA obtained from fixed cells using current methodology typically has an RNA integrity number (RIN) between 1.0 and 4.0, where RIN is based on a scale from 1.0-10.0, with 1.0 referring to totally degraded RNA and 10.0 referring to totally intact RNA.
  • RIN RNA integrity number
  • the methods and kits provided herein represent improvements over existing technology.
  • the methods of this disclosure surprisingly yet reproducibly result in isolated RNA from fixed cells having a RIN of greater than 8.0, and often greater than 9.0.
  • the methods of this disclosure differ from prior art methods in the length and temperature of certain incubations.
  • conventional methods typically require high temperature (e.g., 80°C), limited duration incubation of a fixed biological sample.
  • high temperature incubations were considered necessary in the art to reverse cross-linking induced by fixation.
  • these conventional methods as described herein, generally produce samples of fragmented RNA.
  • certain aspects of the methods provided herein utilize lower temperatures (e.g., about 56°C) for prolonged periods and thereby produce high yields of intact RNA, without the need for additional high temperature (e.g., 80°C) incubations.
  • the method and kits described herein may also be used to isolate RNA from small numbers of cells, even single cells. Using such high quality, intact RNA in downstream research and clinical applications can, inter alia, reduce measurement bias, improve detection and mapping accuracy, and enable quantification of expressed RNA transcripts from fixed cells and tissues.
  • the methods of this disclosure can be applied at the single cell level. This is the first reported method having this capability. This is a significant advantage because of the single cell nature of many biological applications including but not limited to single cancer cell isolation and analysis, stem cell isolation and analysis, and the like.
  • the methods of this disclosure can be applied to archived samples. These methods can be used to extract high-quality RNA at a later time from properly archived and stored samples. These methods are therefore not dependent on the use of freshly harvested tissue. This is a significant advantage particularly in low-resource clinical or research settings where immediate processing is not available or possible.
  • the methods of this disclosure also provide the ability to deeply profile rare cell populations, as exemplified herein.
  • the methods can be used for transcriptome profiling of sorted cells, including fixed, stained and sorted cells from for example primary tissue such as but not limited to fetal tissue.
  • primary tissue such as but not limited to fetal tissue.
  • such analysis may be performed on single cells such as single neural progenitors and single cortical progenitors such as but not limited to [PAX6+ SOX2+ TBR2+] and [PAX6+ SOX2+ TRB2-] cells.
  • This technique can be used to enrich rare populations such as those identified by an antibody or other binding partner.
  • an infrequent (1%) progenitor population is harvested and analyzed, leading to the discrete populations therein such as for example a roughly 0.4% population and a roughly 0.6% population of the total cells.
  • this disclosure provides a method for purifying RNA from a fixed biological sample, comprising: (a) incubating the sample in an aqueous lysis buffer at a temperature between about 50-60°C for a time between about 2-4 hours; (b) contacting the sample with a DNase for a time sufficient to digest DNA; (c) contacting the sample with a substrate that binds RNA, and (d) eluting bound RNA from the substrate.
  • the fixed biological sample is a formalin-fixed cell sample or a formalin-fixed tissue sample.
  • the fixed biological sample is a formalin- fixed paraffin-embedded (FFPE) sample, and optionally the method further comprises an initial deparaffinization step.
  • FFPE formalin- fixed paraffin-embedded
  • step (a) comprises incubating the sample in the aqueous lysis buffer at a temperature between about 52-58°C. In some embodiments, step (a) comprises incubating the sample in the aqueous lysis buffer at a temperature between about 54-57°C. In some embodiments, step (a) comprises incubating the sample in the aqueous lysis buffer at a temperature of about 56°C +/- 2°C. In some embodiments, step (a) comprises incubating the sample in the aqueous lysis buffer at a temperature of about 56°C.
  • step (a) comprises incubating the sample in the aqueous lysis buffer for about 2-3 hours. In some embodiments, step (a) comprises incubating the sample in the aqueous lysis buffer for about 3-4 hours. In some embodiments, step (a) comprises incubating the sample in the aqueous lysis buffer for about 3 +/- 0.5 hours. In some
  • step (a) comprises incubating the sample in the aqueous lysis buffer for about 3.0 hours. In some embodiments, step (a) comprises incubating the sample in the aqueous lysis buffer at a temperature of about 56°C for about 3.0 hours.
  • step (a) comprises incubating the sample in the aqueous lysis buffer for about 0.5 to 3 hours, including 1-3 hours, 1-2 hours, or about 1 hour.
  • the method does not comprise incubating the sample at a temperature above 60°C. In some embodiments, the method does not comprise incubating the sample at a temperature of about 80°C or higher.
  • the lysis buffer comprises a buffering agent and a detergent.
  • the buffering agent is selected from the group consisting of Trips, Mops, Mes, Hepes, phosphates, borates, carbonates, and combinations thereof.
  • the detergent is selected from the group consisting of non-ionic, cationic, anionic and zwitterionic detergents.
  • the lysis buffer further comprises one or more substances selected from the group consisting of ethanol, chelating agents, nucleophilic reagents, reducing agents, inorganic salts, guanidine salts, pH indicators, and stabilizers.
  • the lysis buffer has a pH in a range of from about 4 to 11, from about 7 to 10, or from about 8 to 9.
  • a proteolytic agent either is added to the mixture in step (a) in addition to the lysis buffer, or a proteolytic agent is already comprised in the lysis buffer, the proteolytic agent being selected from the group consisting of proteases and non-enzymatic proteolytic compounds.
  • the proteolytic agent is selected from the group consisting of proteinase K, trypsin, chymotrypsin, papain, pepsin, pronase, endoproteinase Lys- C, bromocyane, recombinant Bacillus proteases, Lysozyme, and a mixture thereof.
  • RNA once eluted, RNA has an RNA integrity number (RIN) between about 5.0-10.0. In some embodiments, the eluted RNA has a RIN of at least 5.0, at least 5.5, at least 6.0, at least 6.5, at least 7.0, at least 7.5, at least 8.0, at least 8.5, at least 9.0, or at least 9.5. In some embodiments, the method produces an amount of eluted RNA of at least 0.2 ⁇ g, at least 2 ⁇ g, or at least 20 ⁇ g. In some embodiments, the RNA once eluted has a purity of at least 1.8 as assessed by the 260 nm/280 nm ratio. In some embodiments, the RNA once eluted comprises no more than 0.01% or 0.001% (w/w) of DNA.
  • RIN RNA integrity number
  • the fixed biological sample comprises at least 10 5 , at least 10 6 , at least 10 7 , at least 10 s , at least 10 9 , at least 10 10 , at least 10 11 , or at least 10 12 cells.
  • the fixed biological sample comprises embryonic stem cells, pluripotent stem cells, somatic cells, germ cells, or gametes.
  • the fixed biological sample comprises human cells or tissue.
  • this disclosure provides a kit for purifying RNA from a fixed biological sample, comprising an aqueous lysis buffer and instructions, wherein the instructions provide that a fixed biological sample should not be incubated in the lysis buffer at a
  • the instructions provide that a fixed biological sample should not be incubated in the lysis buffer at a temperature of 80°C or higher. In some embodiments, the instructions provide that a fixed biological sample is incubated in the aqueous lysis buffer at a temperature between about 50-60°C, for a time between about 2-4 hours.
  • the lysis buffer comprises a buffering agent and a detergent.
  • the buffering agent is selected from the group consisting of Trips, Mops, Mes, Hepes, phosphates, borates, carbonates, and combinations thereof.
  • the detergent is selected from the group consisting of anionic and zwitterionic detergents.
  • the lysis buffer further comprises one or more substances selected from the group consisting of ethanol, chelating agents, nucleophilic reagents, reducing agents, inorganic salts, guanidine salts, pH indicators, and stabilizers.
  • the lysis buffer has a pH in the range of from about 4 to 11, from about 7 to 10, or from about 8 to 9.
  • the kit further comprises a proteolytic agent selected from the group consisting of proteases and non-enzymatic proteolytic compounds.
  • a proteolytic agent selected from the group consisting of proteases and non-enzymatic proteolytic compounds.
  • the proteolytic agent is selected from the group consisting of proteinase K, trypsin, chymotrypsin, papain, pepsin, pronase, endoproteinase Lys-C, bromocyane, recombinant Bacillus proteases, Lysozyme, and a mixture thereof.
  • the kit further comprises one or more of a DNase, RNA binding substrate, and elution buffer.
  • this disclosure provides a method for purifying RNA from a fixed biological sample, comprising: (a) incubating the sample in an aqueous lysis buffer at a temperature between about 50-60°C for a time between about 0.5-3 hours; (b) contacting the sample with a substrate that binds RNA, and (c) eluting bound RNA from the substrate.
  • the fixed biological sample is a formalin-fixed cell sample or a formalin-fixed tissue sample.
  • the fixed biological sample is a formalin- fixed paraffin-embedded (FFPE) sample, and optionally the method further comprises an initial de-paraffinization step.
  • FFPE formalin- fixed paraffin-embedded
  • step (a) comprises incubating the sample in the aqueous lysis buffer at a temperature between about 52-58°C. In some embodiments, step (a) comprises incubating the sample in the aqueous lysis buffer at a temperature between about 54-57°C. In some embodiments, step (a) comprises incubating the sample in the aqueous lysis buffer at a temperature of about 56°C +/- 2°C. In some embodiments, step (a) comprises incubating the sample in the aqueous lysis buffer at a temperature of about 56°C. In some embodiments, step (a) comprises incubating the sample in the aqueous lysis buffer for about 0.5-2 hours.
  • step (a) comprises incubating the sample in the aqueous lysis buffer for about 1 +/- 0.5 hours. In some embodiments, step (a) comprises incubating the sample in the aqueous lysis buffer for about 1.0 hour. In some embodiments, step (a) comprises incubating the sample in the aqueous lysis buffer at a temperature of about 56°C for about 1.0 hour.
  • the method does not comprise incubating the sample at a temperature above 60°C. In some embodiments, the method does not comprise incubating the sample at a temperature of about 80°C or higher.
  • the lysis buffer comprises a buffering agent and a detergent.
  • the buffering agent is selected from the group consisting of Trips, Mops, Mes, Hepes, phosphates, borates, carbonates, and combinations thereof.
  • the detergent is selected from the group consisting of non-ionic, cationic, anionic and zwitterionic detergents.
  • the lysis buffer further comprises one or more substances selected from the group consisting of ethanol, chelating agents, nucleophilic reagents, reducing agents, inorganic salts, guanidine salts, pH indicators, and stabilizers.
  • the lysis buffer has a pH in a range of from about 4 to 11, from about 7 to 10, or from about 8 to 9.
  • a proteolytic agent either is added to the mixture in step (a) in addition to the lysis buffer, or a proteolytic agent is already comprised in the lysis buffer, the proteolytic agent being selected from the group consisting of proteases and non-enzymatic proteolytic compounds.
  • the proteolytic agent is selected from the group consisting of proteinase K, trypsin, chymotrypsin, papain, pepsin, pronase, endoproteinase Lys- C, bromocyane, recombinant Bacillus proteases, Lysozyme, and a mixture thereof.
  • the method further comprises evaluating the RNA integrity of the
  • RNA by gene expression analysis of housekeeping genes or other markers that are consistent with that observed in live cells RNA by gene expression analysis of housekeeping genes or other markers that are consistent with that observed in live cells.
  • the method produces an amount of eluted RNA of at least 0.1 pg, at least 1 pg, at least 10 pg, at least 100 pg, or at least 1000 pg.
  • the fixed biological sample comprises 1-10 cells, or at least 10, at least 50, at least 100, at least 10 3 , at least 10 4 , or at least 10 5 cells. In some embodiments, the fixed biological sample comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cells. In some embodiments, the fixed biological sample comprises embryonic stem cells, pluripotent stem cells, somatic cells, germ cells, or gametes. In some embodiments, the fixed biological sample comprises human cells or tissue.
  • Figures 1A-1E show high quality and yield RNA isolated from fixed and sorted cells.
  • Figure 1A presents representative bioanalyzer traces showing column purified total RNA from 10,000, 1,000, and 100 live or fixed, permeabilized and DAPI-stained HI hESCs with or without reverse-crosslinking (Fixed prep, and Live prep respectively). Data are from two independent experiments. Samples were diluted or concentrated so 66 cell equivalents were loaded per lane.
  • Figures IB and 1C show RNA integrity and RNA yield quantified from HI hESCs.
  • the first three columns of each graph correspond to live cells using the live cell preparation; the second three columns correspond to fixed cells using the live cell preparation; the third three columns correspond to live cells using the fixed cell preparation; the last three columns correspond to fixed cells using the fixed cell preparation.
  • Figure ID shows a comparison of 30-Ct from 82 genes with 11 and 12 replicates of 10,000 live or fixed hES cell samples (respectively) processed with the fixed cell protocol.
  • Figure IE shows a comparison of ACt from qRT-PCR data between total brain RNA (white rows; second, fourth and sixth rows from the top), and fixed HI hESCs (grey shaded rows; first, third, and fifth rows from the top) purified using RNeasy columns or dT25 beads. Data combined from 3 biological replicates.
  • Figures 2A-2F show single cell RNA-Seq from fixed cells.
  • Figure 2A presents representative Bioanalyzer traces of SmartSeq2 amplified cDNA from single hESCs (Live or Fixed) processed by the standard protocol (Live) or with reverse crosslinking and purification using DYNABEADS® 01igo(dT) 2 5 (Fixed).
  • Figure 2B shows cDNA quantity measured after 19 cycles of SmartSeq2 amplification. Data are from two independent experiments except the ERCC only and empty well controls. Error bars indicate standard deviation, number of single cell samples are indicated on bars. Data are from two biological replicates.
  • Figure 2C shows HiSeq RNA-Seq data for each cell processed by the four listed conditions.
  • FIG. 2D shows the number of unique genes detected by RNA-Seq per cell.
  • Figure 2E shows a heatmap with hierarchical clustering for single hES cells showing pairwise correlation. Column colors indicate experimental replicate and rows indicate protocol.
  • Figure 2F presents a table showing number of differentially expressed genes between the different conditions calculated by DESeq.
  • Figures 3A-3D show prospective isolation and profiling of single differentiating hES cells shows concordance between protein expression and gene expression.
  • Figure 3A depicts a sorting schematic showing differentiated single cells sorted from undifferentiated cells using PAX6 intracellular antibody staining. Six single PAX6+ and PAX6- cells were sorted and processed for RNA-Seq by the SmartSeq2 protocol. The PAX6- cells within the region of the trace indicated by "P6" are collected in tubes 1-6, depicted below. The PAX6+ cells within the region of the trace indicated by "P5" are collected in tubes 7-12, depicted below.
  • Figure 3B presents normalized MiSeq RNA-Seq data showing substantial expression of three common housekeeping genes from each sorted cell.
  • Data for each of the cells (lcpos l-lcpos6 and lcnegl-cneg6) consists of three columns: on the left, GAPDH; in the middle, ACTB; and on the right, PPIA.
  • Figure 3C presents normalized MiSeq RNA-Seq data showing population- specific expression of PAX6+ and POU5F1.
  • Data for each of the cells (lcpos l- lcpos6 and lcnegl- cneg6) consists of two columns: on the left, PAX6 and on the right, POU5F1.
  • Figure 3D shows substantial POU5F expression is only highly detected in three of the six PAX6- cells.
  • Figure 4 provides representative BioAnalyzer profiles of RNA extracted according to the methods of this disclosure (left panel) versus control (right panel).
  • Figures 5A-B show that human cortical progenitors are diverse and intermixed during development.
  • Figure 5A Model of the progenitor compartment shows a mixture of radial glial cells (RG), outer RGs (oRGs), intermediate progenitors (IPCs) and other unmarked cortical cell types. Known markers for each cell type are shown below. RGs identified by antibody staining are called SP (SOX + ,PAX + ,TBR2 ⁇ ), and IPCs are called SPT (SOX + ,PAX + ,TBR2 + ).
  • Figure 5B Immunocytochemistry images of 18 PCW germinal zones.
  • Top left Low magnification images stitched together ⁇ left), or individual micrograph ⁇ right) of the VZ, the iSZ, and oSZ stained by DAPI, TBR2, PAX6, and SOX2 with the colors indicated. Scale bars are ⁇ . High magnification for VZ/iSZ region (middle) and oSZ region (bottom) show SP (arrow with filled arrowhead) and SPT (arrowhead) cells. Many cells the VZ, iSZ, and oSZ lack progenitor markers and are unknown cell types (arrows with empty arrowhead). Colors are as in top, scale
  • Figure 6 depicts a schematic for an experiment comparing live and fixed cells and preparations.
  • Figures 7A-7G FSC-Seq of human cortical progenitors identifies genetic signatures for vRGs and oRGs.
  • Figure 7 A Schematic shows single-cell transcriptomic profiling of primary SP and SPT cells. Color scheme is followed throughout Figure, and scale is 1cm.
  • Figure 7B FACS plots show gating strategy and frequency of gated cells compared to total cortical cells. Left plot includes only Go-Gi cortical cells and right plot shows only cells gated on the left plot.
  • Figure 7D
  • FIG. 7E Heatmap showing the correlation of individual fixed singles to the indicated module eigengenes. Module 3 and 4 eigengenes are enriched in mostly non- overlapping sets of SP cells
  • Figure 7F Heatmap shows correlation of single cell-derived eigengenes with 15 PCW (top) and 21PCW (bottom) gene expression data from the BrainSpan Atlas of the Developing Human Brain . Abbreviations are: subgranular zone (SG), subplate (SP), intermediate zone (IZ), outer subventricular zone (oSZ), inner subventricular zone (iSZ), and VZ (ventricle zone).
  • Figure 7G Heatmap showing normalized gene expression of genes differentially expressed between modules vRG and oRG in the single cells (left) and in 21 PCW human tissues (right). Both in the single cell data and the LCM data, vRG and oRG genes largely do not overlap cells or regions. All heat maps show normalized expression with red indicating high expression, blue indicating low expression; and single cell heatmaps in Figure 7E and 7G are clustered as in Figure 7D.
  • Figures 8A-I shows confirmation of markers for vRGs and oRGs, and model for the lineage progression of radial glial progenitors.
  • Figure 8A DESeq analysis of vRG versus oRG single cell data show that HOPX and ANXA1 (in red) are two of the most differentially expressed genes between these groups. All genes with pval ⁇ 0.001 and Log 2 fold change differential expression of >2 or ⁇ -2 are shown.
  • FIG. 8B Greyscale (left) micrographs of the entire cortical depth showing DAPI, SOX2, and either vRG marker ANXA1 or oRG marker HOPX identified from gene expression, and co-stained (right) micrographs of only the germinal zone showing co- staining SOX2 (red) and ANXA1 or HOPX (green).
  • Tissue is 16 PCW cortex, scales are ⁇ .
  • ANXA1 staining overlaps with SOX2 only in the VZ while HOPX bright cells extensively co-stain with SOX2 in the SZ but rarely in the VZ.
  • FIG. 8C Micrograph of germinal zone of 19PCW cortex showing co-staining SOX2 (red) and HOPX (green), scales are lOOpm.
  • Figure 8D High resolution images of ANXA1 (top) and HOPX (bottom) in the oSZ (left) and VZ/iSZ region (right). Scale is 50 ⁇ . Examples of co-expression with SOX2
  • FIG. 8E High resolution micrograph of a mitotic cell in the oSZ co-stained with SOX2, HOPX and phospho-VIM. Scale bar is 20 ⁇ .
  • Figures 8F-H Quantitation of SOX2 + ,HOPX + or HOPX " cells in oSZ that are Ki67 + (f); percentage of SOX2 + cells that are also HOPX + ( Figure 8G); and the percentage of HOPX + cells that are also SOX2 + ( Figure 8H). Data is mean +/- St. Dev.
  • Figure 9 shows high quality and yield RNA from fixed and sorted cells. Total recovered RNA yield quantified from indicated numbers of HI hESCs. Error bars indicate standard deviation. Sample names are described previously ( Figure 6), sample numbers were as in Figure IB. Quantities for 100 cell samples are not shown since low molecular weight broad peak that appeared after concentration (not RNA specific) interfered with quantitation.
  • Figures 10A- 10B show single cell FSC-Seq with hESCs.
  • Figure 10A Bar plot showing the number of mapped reads per cell. Single-cell libraries were sequenced to a moderate depth (7.0M reads +/- 0.9M). 100% is all barcoded reads independent of mapping.
  • Figure 10B Alignment statistics for HiSeq RNA-Seq data for cells processed by the four listed conditions. * p ⁇ 0.05 and ** p ⁇ 0.001 by homoscedastic TTEST.
  • Figures 11 A- 1 IB shows gating strategy for prospective isolation and profiling of human cortical progenitor cells.
  • Figure 11 A A representative image of the entire gating strategy for the isolation of the SP and SPT cells. Top row: cells are gated for morphology, then single cells, and last for the presence of DAPI. Only cells in the Go-Gi phase of the cell cycle were sorted. Bottom row: Progenitors were first identified by high expression of SOX2 and PAX6, and then for the presence (orange) or absence (blue) of TBR2. Bottom right: Projection of progenitor populations on all cells gated by the strategy on the top row and stained for TBR2 and DCX show that SP and SPT progenitors are negative for DCX.
  • Figures 12A-12F show MiSeq read statistics for human prenatal SP and SPT cells.
  • FIG. 12A Representative Bioanalyzer traces show cDNA amplification of one SP and one SPT cell.
  • Figure 12B Histogram showing the distribution of SP and SPT cells with respect to the number of mapped reads. Cells with fewer than 50,000 mapped reads were excluded (average 119,252 + 55,746 reads).
  • Figure 12C Mapping percent for each cell with more than 50,000 mapped reads; 100% is all barcoded reads independent of mapping.
  • Figure 12D Correlation between cells based on high variability genes and
  • Figure 12E all ERCCs reads only. High variability genes but not the ERCCs (spiked into each sample at defined
  • concentrations segregate cells into blocks based on the cell phenotype of the sorted cell.
  • Figure 13 shows data from human cortical progenitors prepared by FSC-Seq clustered appropriately with independent data from live single cells.
  • Data from fixed SP and SPT cells were compared to an existing data set generated from live prenatal human neuronal single cell by hierarchical clustering.
  • Clustering was based on the common 482 of the top 500 genes contributing to the first principal component that was identified in a previous study 13.
  • the two previously published IPCs cluster with the fixed SPT cells, and previously published RGs cluster with our fixed SP cells. This clustering robustly categorizes cells by cell type despite the two data sets differing in: sample age, sample donor, dissociation, cell capture, cDNA amplification, and using live or fixed cells.
  • NPCs are neural precursor cells generated from hESCs 13. Lower color bar annotations were based on annotation from the earlier study 13.
  • Heatmaps of the genes that comprise each of the 6 WGCNA modules are shown. Heatmap colors show gene expression normalized between cells of a given gene. Red indicates high expression, blue indicates low expression. SP and SPT cells are noted by the color bar and in the legend, and are organized based on clustering of WGCNA eigengenes (Figure 7D).
  • Figure 16 shows modules 3 and 4 are enriched in non-overlapping sets of SP cells.
  • Figures 17A-17C Heatmaps show the enrichment of the module eigengene expression pattern on Allen Brain Atlas Human prenatal cortical gene expression data. Regions that correlate better to any given eigengene are red, while poorer correlation is blue.
  • Figure 17B Heatmaps shows specific marker genes expressed in the BrainSpan Atlas of the Developing Human Brain . Genes were differentially-expressed between module 3 and 4 enriched cell, though they may not be in the original modules ( Figure 8A). Regions with strong correlation to a given gene are red, while poor correlation is blue.
  • Figure 17C Expression of HOPX in macaque though development. Plot shows expression of HOPX in the visual cortex (VI) of the NIH Blueprint Non-Human Primate Atlas. Each column shows different ages from embryonic day 40 (E40), through 48 months after birth (48M). Rows are micro-dissected regions. Red color indicates high expression. Abbreviations for prenatal time points are:
  • MZ marginal zone
  • oCP outer cortical plate
  • oCP inner cortical plate
  • SP subplate
  • IZ intermediate zone
  • iSZ outer subventricular zone
  • iSZ inner subventricular zone
  • VZ ventilation zone
  • Abbreviations for postnatal time points are: Layer 1-6 (L1-L6), and white matter (WM).
  • HOPX is initiated in VI at E70, when the oSZ is formed, and while first detected in the entire germinal zone, is subsequently enriched in the oSZ.
  • Figure 18 expression of HOPX is initiated in VI at E70, when the oSZ is formed, and while first detected in the entire germinal zone, is subsequently enriched in the oSZ.
  • SP/IZ subplate/intermediate zone
  • OSZ outer subventricular zone
  • iSZ inner subventricular zone
  • VZ ventricle zone
  • Figure 19 shows greyscale and merged micrographs of a representative region of oSZ stained for SOX2, HOPX, and Ki67.
  • White arrows are SOX + , HOPX " , Ki67 + cells and arrowheads are SOX + , HOPX + , Ki67 + cells.
  • Fixation is a chemical process by which cells and tissues are preserved from decay, thereby preventing autolysis or putrefaction. Fixation terminates any ongoing biochemical reactions, and may also increase the mechanical strength or stability of the treated cells or tissues. Fixation theoretically preserves cells and tissues as close to their natural state as possible. This is desirable if the cells or tissues are to be examined, such as by gene expression and histological studies.
  • a common fixative agent is formaldehyde, which is typically employed as a saturated water solution that contains about 40% formaldehyde by volume or 37% by mass (referred to as "100% formalin").
  • Formalin is a commercial name for
  • formaldehyde i.e., the active fixative in formalin is formaldehyde.
  • Formaldehyde preserves (or fixes) cells or tissues by inducing cross-linking of primary amino groups in proteins with other nearby nitrogen atoms in proteins or nucleic acids. While formaldehyde fixed cells or tissues are preserved for analysis (which can be performed immediately or even years later), the molecular cross-linking that underlies fixation is believed to cause significant challenges to certain applications, including for example to studies involving ribonucleic acids (RNA), such as messenger RNA (mRNA) and microRNA (miRNA).
  • RNA ribonucleic acids
  • mRNA messenger RNA
  • miRNA microRNA
  • the fixation process typically results in the isolation of heavily fragmented and chemically (e.g.,
  • RNAs that are not accurately reflective of the natural RNA pool that existed in the cell just prior to fixation.
  • these degraded fragments of RNA can be biased (e.g., unrepresentative of true abundance levels in the cell), difficult to identify (e.g., when fragments are short or map to multiple regions), and difficult to quantify (e.g., due to incomplete or biased recovery and lack of mapping confidence).
  • RNA quantity and quality is typically classified according to its purity, concentration, and the relative distribution of fragment sizes (reviewed by Fleige and Pfaffl, "RNA integrity and the effect on the real-time qRT-PCR performance.” Mol Aspects Med. 2006 Apr-Jun;27(2- 3): 126-39).
  • Spectrophotometric methods such as Nanodrop (Thermo Fisher Scientific, Waltham, Massachusetts) and Qubit (Life Technologies, Carlsbad, California), measure the concentration of nucleic acids and their purity, or the contribution of nucleic acids relative to non-nucleic acids to the absorbance spectrum, but generally do not measure RNA integrity (e.g., quality of RNA and/or the intact nature of the RNA). Determination of RNA integrity may be performed by measuring the distribution of RNA fragment sizes using the capillary
  • RNA to migrate through a microfluidic channel is proportional to fragment size. Longer fragments take a longer time to migrate and be detected. The abundance of fragments present is inferred from the intensity of a fluorescent dye that binds nucleic acids.
  • RNA measured on a Bioanalyzer produces a size distribution profile characterized by two abundant and narrow peaks at high molecular weight representing the 28S (larger) and 18S ribosomal RNA fragments (see RIN 10 from Figure 2 of Schroeder et al., "The RIN: an RNA integrity number for assigning integrity values to RNA measurements.” BMC Molecular Biology. 2006, 7:3., reproduced below).
  • the lower molecular weight peaks apparent as small jagged peaks to the left of the 18S and 28S peaks are indicative of RNA degradation and contribute to lower RIN scores.
  • RNA integrity number is calculated based on the peak profile of the electropherogram trace using an algorithm designed by Agilent for use with the Bioanalyzer (Schroeder, 2006). RINs range from 1 (degraded RNA) to 10 (intact RNA). Below is shown the overall size distribution, characteristic rRNA peaks (see large peaks in RIN 10 plot), and RINs for a range of good to poor quality RNA samples, as reported by Schroeder et al. From these plots, it is apparent that higher RIN samples have distinctive 28S and 18S peaks and that these peaks diminish in lower RIN samples and lower molecular weight peaks become more apparent.
  • RNA from formaldehyde fixed cells The industry standard to isolate RNA from formaldehyde fixed cells is to use a commercially available kit, such as those provided by Qiagen (Hilden, Germany; e.g., miRNeasy FFPE RNA Isolation Kit, cat. no. 217504), Life Technologies (Carlsbad, California; e.g., RecoverAllTM Total Nucleic Acid Isolation Kit, cat. No. AM1975), Agilent Technologies (Santa Clara, California), NuGEN Technologies (San Carlos, California), and Promega
  • RNA e.g., mRNA
  • oligo-dT primers to amplify cDNA from all mRNA in an unbiased fashion (due to intact mRNAs having a poly-A tail).
  • fragmented mRNA will not be consistently amplified due to loss of the poly-A tail.
  • certain manufacturers recommend using gene-specific primers or random nonamers to amplify the fragmented RNA generated according to their methodology, as opposed to conventional oligo-dT primers employed with intact RNA.
  • the methods described herein which provide for isolation of intact RNA from fixed cells having a RIN comparable to that of RNA obtained from fresh (un-fixed) cells, allow for and enable the use of the RNA without the need for further modification or processing, as is common with fragmented RNA. This is desirable, as it reduces potential bias associated with methods for amplifying fragmented RNA.
  • RNA having RINs corresponding to a sample of RNA having largely intact RNA e.g., RIN > 8.0
  • the fixed biological sample is any biological sample containing RNA, which was fixed, e.g., subjected to a fixation condition.
  • Such fixed biological samples include fixed cells including embryonic stem cells, pluripotent stem cells, somatic cells, germ cells, and gametes.
  • the fixed biological sample is a fixed tissue, such as connective tissue (e.g., areolar tissue, reticular tissue, adipose tissue, bone, cartilage, blood), epithelial tissue (e.g., simple epithelial tissue, secretory epithelial tissue), muscle tissue (e.g., skeletal muscle tissue, smooth muscle tissue, cardiac muscle tissue), and nervous tissue (e.g., brain tissue, spinal cord tissue, peripheral nervous tissue).
  • connective tissue e.g., areolar tissue, reticular tissue, adipose tissue, bone, cartilage, blood
  • epithelial tissue e.g., simple epithelial tissue, secretory epithelial tissue
  • muscle tissue e.g.,
  • the cells or tissue are obtained from a subject, such as a human subject.
  • the subject is a non-human mammal.
  • the subject is a non-human primate.
  • the subject is a rodent.
  • the subject is a sheep, a goat, a cattle, a cat, or a dog.
  • the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode.
  • the subject is a research animal.
  • the subject, cells or source of the cells is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either sex and at any stage of development.
  • fixative agent such as formaldehyde (commercially available as "formalin”).
  • fixative agents include, but are not limited to, para-formaldehyde, glutaraldehyde, methanol, mercurials (e.g., B-5 and Zenker's fixative), picrates, and HOPE fixative (Hepes-glutamic acid buffer-mediated organic solvent protection effect fixative).
  • the methods comprise incubating a fixed biological sample with an aqueous lysis buffer ("lysis buffer").
  • lysis buffer is any buffer that lyses the fixed cells and tissues, thereby releasing the RNA within.
  • aqueous lysis buffers are known in the art, and include, for example, those commercially available from vendors such as Qiagen: Buffer PKD (Proteinase K digest buffer, cat. no. 1034963); Life Technologies: Digestion Buffer (e.g., from RecoverAllTM Total Nucleic Acid Isolation Kit, cat. No. AM1975), Agilent Technologies: Proteinase K
  • Digestion Buffer e.g., from absolutely RNA FFPE Kit, cat. no. 400809; and Promega: Lysis Buffer (LB A, cat. no. Z101A).
  • Other lysis buffers include those described in U.S. published patent applications US 2013/0280787, US 2013/0280728, and US 2013/0164825, the entire contents of each are hereby incorporated by reference.
  • lysis buffer comprises a buffering agent and a detergent.
  • Buffering agents are well known, and include, without limitation, Trips, Mops, Mes, Hepes, phosphates, borates, carbonates, and combinations thereof.
  • Detergents are also well known, and include, without limitation, non-ionic, cationic, anionic and zwitterionic detergents.
  • the detergents are selected from anionic or zwitterionic detergents.
  • the lysis buffer may comprise anionic detergents, such as sodium dodecyl sulfate (SDS).
  • nonionic surfactants such as substituted phenol or sugar polyethoxylates, commercially available for instance as Triton X-114 (Dow Chemical Co., Midland, Mich., USA), Triton X-100 (Dow Chemical Co., Midland, Mich., USA) or Tween 20 (Merck, Darmstadt, Germany) may be used, as well as cationic surfactants, such as quaternary ammonium surfactants, e.g. cetyl trimethylammonium bromide (CTAB) or tetradecyl trimethylammonium bromide (TTAB).
  • CTAB cetyl trimethylammonium bromide
  • TTAB tetradecyl trimethylammonium bromide
  • the lysis buffer comprises a proteolytic agent, which digests proteinaceous components of the cells and/or tissue, helping to break down the cellular components and allowing RNA to be released.
  • proteolytic agents are known in the art, and include, without limitation, proteinase K, trypsin, chymotrypsin, papain, pepsin, pronase, endoproteinase Lys-C, bromocyane, recombinant Bacillus proteases, and lysozyme.
  • the lysis buffer comprises proteinase K.
  • the proteolytic agent e.g., proteinase K
  • the proteolytic agent is not comprised in the buffer, but is added to the sample, e.g., after the lysis buffer has been added to the sample.
  • the lysis buffer further comprises one or more of ethanol, chelating agents, nucleophilic reagents, reducing agents, inorganic salts, guanidine salts, pH indicators, and stabilizers.
  • the lysis buffer comprises a nucleophilic reagent, such as those specifically provided in paragraphs [0040]-[0052] of U.S. published patent application US 2013/0280728.
  • the lysis buffer has a pH in a range of from about 4 to 11, from about 7 to 10, or from about 8 to 9.
  • the lysis buffer comprises guanidine salts.
  • a binding buffer which may be used after proteinase K digestion and cross-linking reversal, comprises guanidine salts.
  • the lysis buffer comprises one or more nucleophilic reagents such as nucleophilic reagents that induce cross-linking reversal. Examples may be found in published US application US 2013/0280728.
  • the method comprises incubating the fixed biological sample in an aqueous lysis buffer at a temperature that is sufficient to free RNA and allow for the isolation of intact RNA.
  • RNA is defined as RNA having a RIN of at least 5.0, at least 5.5, at least 6.0, at least 6.5, at least 7.0, at least 7.5, at least 8.0, at least 8.5, at least 9.0, or at least 9.5.
  • intact RNA has a RIN > 8.0.
  • RNA that, when assessed by gene expression, shows substantial reproducible expression of housekeeping genes.
  • housekeeping genes include, without limitation, GAPDH, ACTB, and PPIA.
  • the methods provided herein do not involve incubating the sample at a temperature above 60°C. In some embodiments, the methods provided herein do not comprise incubating the sample at a temperature above 65°C, 70°C, 75°C, 80°C, or higher. In some embodiments, the methods do not comprise incubating the sample at a temperature of 80°C or higher.
  • the methods comprise incubating a fixed biological sample at a temperature of about 50-60°C. In some embodiments, the methods comprise incubating a fixed biological sample at a temperature of 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, or 60°C. In some embodiments, the methods comprise incubating a fixed biological sample at a temperature of 56°C +/- 2°C. In some embodiments, the methods comprise incubating a fixed biological sample at a temperature of 56°C.
  • the methods further comprise incubating a fixed biological sample in an aqueous lysis buffer for a time and at a temperature that is sufficient to free RNA and allow for the isolation of intact RNA.
  • Conventional methods generally require incubations for times of less than one hour, for example 15 minutes at a temperature between 50-60°C, followed by a brief (e.g., -15 minute) incubation at a higher temperature, for example 80°C.
  • a brief incubation times and temperatures generally produce fragmented isolated RNA.
  • the methods comprise incubating a fixed biological sample in a lysis buffer (e.g., as described herein) for a time of about two (2) to four (4) hours at 50-60°C.
  • the methods comprise incubating a fixed biological sample for a time of about 2-3 hours, 3-4 hours, or for about 3.0 hours +/- 0.5 hours.
  • the methods comprise incubating a fixed biological sample for about 3 hours (e.g., at a temperature as described herein, including a temperature in the range of 50-60°C such as but not limited to 56°C).
  • the methods comprise incubating a fixed biological sample in a lysis buffer (e.g., as described herein) for a time of about 0.5 to 3 hours at 50-60°C. In some embodiments, the methods comprise incubating a fixed biological sample for a time of about 0.5-2 hours, 0.5-1 hours, or for about 1 hour +/- 0.5 hour. In some embodiments, the methods comprise incubating a fixed biological sample for about 1 hour (at a temperature as described herein, including a temperature in the range of 50-60°C such as but not limited to 56°C).
  • the methods further comprise contacting a fixed biological sample with a DNase.
  • DNase enzymes and other enzymes affecting e.g., non-target biomolecules (RNAs) as well as buffers and solutions that can be used for performing a respective enzymatic treatment are well known in the art.
  • suitable DNases are commercially available from the vendors described herein, and include RNase-free DNase I available from Qiagen (cat. no. 79254).
  • the sample is contacted with a DNase following the incubation in lysis buffer, so as to increase the enzyme's ability to digest DNA that has been released as a result of the lysis/digestion step.
  • an additional buffering agent may be added to increase the efficiency of the DNase treatment (e.g., Qiagen DNase Booster Buffer).
  • the methods further comprise contacting the sample with a substrate that binds RNA.
  • the substrate is referred to as a "membrane,” and is comprised in a column suitable for centrifugation (e.g., in a microcentrifuge).
  • a column comprising a substrate (e.g., membrane) that binds RNA is referred to as a spin column.
  • spin columns are well known in the art, and include those provided by the vendors described herein.
  • suitable RNA binding substrates include the Qiagen RNEASY® MINELUTE® Spin Columns (e.g., from the miRNeasy FFPE RNA Isolation kit, cat. no.
  • the substrate is referred to as a "bead” and binds to nucleic acid (e.g., RNA).
  • RNA nucleic acid
  • Such beads may be separated from a solution by centrifugation, filtration, use of a magnet, or any other method known in the art.
  • Beads compatible for use in the methods described herein include those provided by the vendors described herein.
  • suitable RNA binding beads include, without limitation, DYNABEADS® 01igo(dT) 2 5 beads from Life Technologies (e.g., Life Technologies cat. no. 61002) and MagJET Magnetic Beads from Thermo Scientific (e.g., from MagJet RNA Kit, cat. Not. #K2731).
  • the beads may be washed and then are added to a sample.
  • the beads and bound RNA are separated from the sample, for example by centrifugation, and the supernatant containing contaminants and other undesired components is removed.
  • binding buffer is added to the sample to enhance the binding of the RNA to the substrate.
  • binding buffers are well known in the art, and include those contained in commercially available kits, such as those described herein.
  • the method further comprises eluting the RNA.
  • Methods and reagents for eluting bound RNA from an RNA-binding substrate are well known.
  • RNA may be eluted from an RNA-binding substrate using water (e.g., RNase-free water).
  • RNA may be eluted using a commercially available elution buffer, such as those provided in commercially available kits described herein.
  • ethanol is added to the sample prior to eluting the RNA.
  • the substrate prior to eluting the RNA, and after liquids have been spun through (out) of the column, the substrate is washed to remove contaminants, for example using a commercially available wash buffer, such as those contained within commercially available kits described herein.
  • a commercially available wash buffer such as those contained within commercially available kits described herein.
  • the RNA is eluted by contacting the substrate (e.g., membrane) with water or an elution buffer, and centrifuging the column so as to pass the water or elution buffer through the substrate, thereby removing the substrate.
  • RNA from the substrate and collecting the RNA in a collection tube Once eluted, the integrity of the RNA can be assessed, as described herein.
  • the methods and kits described herein may be used to isolate high quality RNA from biological samples that contain any cell or cell type known in the art.
  • cells include, without limitation, embryonic stem cells, pluripotent stem cells, somatic cells, germ cells, gametes, or a combination thereof.
  • the biological sample may contain at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , or at least
  • the methods and kits may be used to isolate high quality RNA from biological samples that contain a small number of cells or even a single cell.
  • the biological sample may contain 1-10 cells, or at least 10, at least 50, at least 100, at least 10 4 , or at least 10 5 cells.
  • the biological sample may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 cells.
  • the cells are from fetal tissue, including human fetal tissue. Kits
  • kits for isolating RNA comprise reagents necessary for performing the methods as provided herein.
  • the kit comprises an aqueous lysis buffer, e.g., as provided herein.
  • the lysis buffer comprises a buffering agent and a detergent, as provided herein.
  • the lysis buffer further comprises one or more substances selected from the group consisting of ethanol, chelating agents, nucleophilic reagents, reducing agents, inorganic salts, guanidine salts, pH indicators, and stabilizers.
  • the lysis buffer has a pH in the range of from about 4 to 11, from about 7 to 10, or from about 8 to 9.
  • the kit comprises a proteolytic agent selected from the group consisting of proteases and non-enzymatic proteolytic compounds.
  • the proteolytic agent is selected from the group consisting of proteinase K, trypsin, chymotrypsin, papain, pepsin, pronase, endoproteinase Lys-C, bromocyane,
  • the kit comprises one or more of a DNase, RNA binding substrate, and elution buffer.
  • the kit typically comprises instructions.
  • the instructions may be written on one of the containers of the kit, including for example a box or other housing within which all components are contained. Alternatively, the instructions may be written on paper or other tangible form provided with the kit.
  • the instructions provide that a fixed biological sample should not be incubated in the lysis buffer at a temperature above 60°C, above 65°C, above 70°C, above 75°C, or above 80°C. In some embodiments, the instructions provide that a fixed biological sample should not be incubated in the lysis buffer at a temperature of 80°C or higher. In some embodiments, the instructions provide that a fixed biological sample is incubated in the aqueous lysis buffer at a temperature between about 50-60°C, for a time between about 2-4 hours. In some embodiments, the instructions provide that a fixed biological sample is incubated in the aqueous lysis buffer at a temperature between about 50-60°C, for a time between about 0.5-3 hours.
  • the instructions provide that a fixed biological sample is incubated in lysis buffer at a temperature of 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, or 60°C. In some embodiments, the instructions provide that a fixed biological sample is incubated in lysis buffer at a temperature of 56°C +/- 2°C. In some embodiments, the instructions provide that a fixed biological sample is incubated in lysis buffer at a temperature of 56°C. In some embodiments, the instructions provide that a fixed biological sample is incubated in lysis buffer for a time of about 2-3 hours, 3-4 hours, or for about 3.0 hours +/- 0.5 hours.
  • the instructions provide that a fixed biological sample is incubated in lysis buffer for a time of about 0.5-2 hours, 0.5-1 hours, or for about 1 hour +/- 0.5 hour. In some embodiments, the instructions provide that a fixed biological sample is incubated in lysis buffer for about 3 hours (e.g., at about 56°C). In some embodiments, the instructions provide that a fixed biological sample is incubated in lysis buffer for about 1 hour (e.g., at about 56°C).
  • the materials used are commercially available from Qiagen, as part of the miRNeasy FFPE RNA Isolation kit (Cat# 217504).
  • the materials include:
  • the cells used in this example were mouse embryonic stem cells that were formaldehyde fixed, permeabilized, intracellular antibody stained, and FACs sorted prior to isolating RNA according to the protocol below.
  • Mouse embryonic stem cells were differentiated to obtain populations enriched for early germ layer progenitor cell types as well as intermediate cell types, as defined by the presence and/or absence of a combination of transcription factor markers.
  • ES cells were maintained in LIF+2i (Ying et al., Nature 2008)
  • Differentiated populations were obtained by manipulation of Fgf, Wnt and/or Bmp signaling for 1 to 5 days
  • cells were trypsinized with 0.01% trypsin, then quenched with FBS and collected in a 15mL conical tube.
  • Cells were washed three times with lmL PBS containing RNasin, after which they were permeabilized with lmL of ice-cold 100% methanol and transferred to 1.5mL non-stick tubes.
  • cells were resuspended in primary antibody solution at a density of -10 cells/mL and incubated overnight at 4°C with gentle agitation.
  • the staining targets were primarily transcription factors to allow for identification and FACS -purification of different cell types found within heterogeneous populations.
  • Cells were transferred to secondary antibody solution at a density of -10 cells/mL and incubated for 1 hour in the dark at 4°C with gentle agitation.
  • Cells were transferred to fresh FACS buffer containing 2U/uL RNasin at a density of -10 cells/mL and sorted via FACS (BD FACS Aria) into 1.5mL tubes pre-coated with FACS buffer to minimize sticking and containing ⁇ to 500 ⁇ FACS buffer with 2U/uL RNasin. Protocol
  • the protocol in this Example, represents an improvement over the protocol
  • the improved method allowed microgram quantities of RNA having a RIN >9.0 to be obtained.
  • DNase Buffer Booster equivalent to a tenth of the total sample volume (approximately 16 ⁇ ) and 10 ⁇ DNase I stock solution was added to the sample. The sample was mixed gently by inverting the tube.
  • Step 10 was repeated until the entire sample had passed through the spin column.
  • the spin column was then placed in a new 2 ml collection tube, and was centrifuged at full speed for five (5) minutes. 15) The spin column was then placed in a new 1.5 ml collection tube. 20 ⁇ of RNase-free water was added directly to the spin column membrane. The sample was then centrifuged at full speed for one (1) minute to elute the RNA.
  • RNA quality was RIN -9.2+1.2 (mean + standard deviation), as compared to RIN of -6.2 + 2.8 for RNA that was not extracted according to this protocol.
  • Dissociated, live, cultured, human cells were in single-cell suspension in lxPBS within either a 15ml conical tube (>1M cells) or a 1.7ml tube ( ⁇ 1M cells). All steps other than centrifugation were performed in an RNase-free hood (Airclean600, AirClean Systems). Cells were centrifuged for 3min at 335xG to pellet then the supernatant was aspirated. The cells were resuspended in 4% PFA in PBS (0.5ml per million cells) and incubated on ice for 15 minutes. The cells were pelleted at 524xG and supernatant removed. The cells were washed one time in 2ml Staining Buffer (SB). Aliquots of two million cells were then resuspended in SB (200 ⁇ per 2 million cells) and stored at -80°C in 1.7ml tubes.
  • SB Staining Buffer
  • CS Capture Solution
  • Samples of more than 1000 cells were not tested with the dT25 bead purification system described below. These samples may be purified by starting at step 10 of "Purification of Total RNA from microdissected FFPE Tissue Sections" protocol from the miRNeasy FFPE Kit (Qiagen - 217504).
  • Cells in CS were thawed by hand warming, vortexed for 5 sec at maximum speed, and spun down quickly without forming a pellet for -lsec in microcentrifuge. The cells were then incubated 1 hour at 56°C in a thermal cycler.
  • ⁇ of dT25 beads were washed 3 times in lx Hybridization Buffer (HB). The beads were finally resuspended in 2xHB in half of the original volume to create a 2x bead concentrate.
  • HB Hybridization Buffer
  • the beads were then washed three times in ⁇ lxHB then once in ⁇ PBS. These washes were performed by resuspending the beads by flicking, centrifuging the tubes, and then using a plate magnet (Life Technologies) to pellet the beads. After the last wash all excess PBS was removed from the beads by pipet. Then 2.8 ⁇ 1 of RNase free water was added. The beads were resuspended by gentle flicking while avoiding the liquid sticking to the sides and cap. The tubes were immediately incubated at 80°C in a thermal cycler with a 90°C hot lid for 2 minutes. The contents of the tubes were quickly spun down in a microcentrifuge, placed on the magnetic rack, and the mRNA containing supernatant was transferred to a clean PCR tube. The pure mRNA was stored at -80°C. Results
  • RNA was purified from 1, 10 or 100 HI hES cells or 10, 100, or lOOOpg of Total Brain (TB) RNA using both methods, and then compared by qRT-PCR ( Figure IE).
  • Figure IE qRT-PCR
  • HI hESCs would be suitable for genome wide transcriptional profiling.
  • Single live, or fixed permeablized and DAPI- stained cells were subjected to full length polyadenylated transcript amplification using the SmartSeq2 method (Picelli, Bjorklund et al. 2013).
  • cells were either applied directly to the cell lysis buffer, or were reverse crosslinked and mRNA purified with dT25 beads. Comparable amounts of cDNA were amplified in all conditions except for fixed cell sorted directly into the cell lysis buffer
  • RGs radial glial progenitor cells
  • RGs can be classified into ventricle zone (VZ)-enriched RGs (vRGs) and outer subventricular zone (oSZ)-localized RGs (oRG).
  • VZ ventricle zone
  • vRGs ventricle zone
  • oSZ outer subventricular zone
  • oRG outer subventricular zone
  • HOPX a tumor suppressor that can inhibit immediate early genes (IEGs) specifically expressed in vRGs.
  • IEGs immediate early genes
  • Tissue was provided by the birth Defects Research Laboratory (BDRL) at the University of Washington, who obtained appropriate written informed consent and provided available non- identifying information for each sample. Cortical pieces were divided into one half for sectioning and the other half for cell isolation. The half for sectioning was fixed in 4% PFA in PBS overnight at 4°C, then cryoprotected in 30% sucrose in PBS for 48-72 h, rinsed briefly with PBS and embedded and frozen in OCT. The other half (approx. 0.25 - 0.5 mL volume) was minced into small pieces with #5 forceps (Fine Science Tools, Foster City CA) in Ca 2+ - and Mg 2+ -free HBSS (14175-095, Life Technologies, Chicago IL, Chicago IL).
  • Minced pieces were treated with 2 mL trypsin solution for 20 min at 37°C (Ca 2+ - and Mg 2+ -free HBSS, 10 mM HEPES, 0.5 mM EDTA, 0.25 mg/ml bovine pancreatic trypsin (EMD Millipore, Billerica MA), 10 ⁇ g/ml DNase I (Roche, Basel, Switzerland), pH 7.6).
  • Digestion was quenched with 6 mL of ice-cold quenching buffer (440 ml Leibovitz L-15 medium, 50 ml water, 5 ml 1M HEPES pH 7.3-7.4, 5 ml lOOx Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na 2 H 2 EDTA], lg bovine serum albumin [A7030, Sigma, St. Louis MO]) containing 100 ⁇ g/ml trypsin inhibitor (T6522, Sigma) and 10 ⁇ g/ml DNase I (Roche).
  • quenching buffer 440 ml Leibovitz L-15 medium, 50 ml water, 5 ml 1M HEPES pH 7.3-7.4, 5 ml lOOx Pen-Strep, 20 ml 77.7 mM EDTA pH 8.0 [prepared from Na 2 H 2 EDTA], lg bovine serum albumin [A7030, Sigma
  • Samples were then pelleted (220xg, 4 min, 4°C), resuspended with 1 mL of quenching buffer and triturated on ice with a P1000 pipette set to 1 mL, using 25 gentle cycles up and down without forming bubbles.
  • the cell suspension was then diluted to 30-40 mL in quenching buffer, filtered through a 45 micron cell filter, pelleted (220xg, 10 min, 4°C), resuspended in 5 mL Staining Medium, and counted on a hemocytometer (typically -30-50 million live cells isolated per cortical piece at -50% viability).
  • HI hESCs (WiCell, Madison WI) were maintained on Matrigel (Corning) in mTESRl media (StemCell Technologies, Vancouver BC). Adherent cell cultures were dissociated with StemPro Accutase Cell Dissociation Reagent (Life Technologies, Chicago IL). The cells were centrifuged (220xg, 3 min) and the dissociation solution was removed. Cells were washed then resuspended in RNase-free Staining Buffer (SB) (1XPBS pH 7.4, 1% BSA (Gemini)
  • the single cell suspension was fixed with 4% PFA (Electron Microscopy Sciences, Hatfield PA) in PBS on ice for 15 minutes, then pelleted (335xg, 3 min, 4°C), washed once with 1 mL SB, then resuspended in SB at 10 million cells/mL, and frozen at -80°C in aliquots.
  • PFA Electro Microscopy Sciences, Hatfield PA
  • Antibodies used were: Alexa488 or PE-conjugated anti-PAX6 (018-1330; BD Biosciences), PE-conjugated anti-DCX (30/Doublecortin; BD Biosciences), PerCP-Cy5.5-conjugated anti-SOX2 (030-678; BD Biosciences), eFluor660-conjugated anti- EOMES (WD1928; eBiosciences), Alexa647-conjugated anti-OCT3/4 (40/Oct-3; BD
  • Sorting was carried out on a BD FACS ARIA-II SORP (BD Biosciences, San Jose CA) using a 130um nozzle. Single cells were sorted into strip tubes containing 5 ⁇ 1 of PKD Buffer (Qiagen, Germantown MD) with 1: 16 Proteinase K Solution (Qiagen, Germantown MD).
  • PKD/Proteinase K solution were thawed at room temperature (RT), mixed, and incubated at 56°C for 1 h. Samples were then centrifuged at 20,000xg for 20 min. The supernatant was transferred to a new tube and ⁇ of DNase Booster Buffer and ⁇ of DNase I stock solution were added with mixing by inversion. Samples were incubated 15 min at RT then 320 ⁇ 1 of buffer RBC was added.
  • RNA with dT25 beads samples were thawed at RT, mixed, and then incubated at 56°C for 1 h in a thermal cycler with the lid set at 66°C (Bio-Rad, Hercules CA). Cells were vortexed for 10 sec, spun down, and placed on ice. Oligo dT25 magnetic beads (Life Technologies Inc.).
  • Beads were added to reverse crosslinked samples and they were heated to 56°C for 1 min, incubated at RT 10 min to allow mRNA hybridization, then placed on ice. Beads were washed three times in ⁇ of ice cold Hybridization Buffer, followed by a subsequent wash using ice cold IX PBS. All PBS was removed and 2.8 ⁇ 1 of RNase free water was added, the beads resuspended, and the mixture was incubated at 80°C for two min then immediately pelleted on a room temperature magnet. The supernatant containing mRNA was rapidly removed and transferred to a new tube and stored at -80°C.
  • RNA from single hESC or prenatal cells are purified using Ampure XP beads (Beckman Coulter,
  • cDNA was quantified with either High Sensitivity DNA Chip (Agilent, Santa Clara CA) on a Bioanalyzer 2100, or with Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies Inc.
  • RNA-Seq libraries Single cells from primary tissue appeared to contain lower cellular RNA content compared to HI hESCs, requiring a reduction in ERCC spike-in RNAs by 10-fold and addition of three extra PCR cycles.
  • Sequencing was carried out on Illumina MiSeq using 31 base paired-end reads.
  • Raw read data was aligned to GRCh37 (hgl9) using the RefSeq annotation gff file downloaded on 4/23/2013.
  • Transcriptome alignment was performed first using RSEM 27 , unmapped reads were then aligned to hgl9 using Bowtie 28 , and remaining unmapped reads were aligned to the ERCC sequences.
  • mRNA mitochondrial RNA
  • mRNA mitochondrial RNA
  • mRNA, and rRNA noncoding RNA
  • genome and ERCC only.
  • Principle component and clustering analysis were performed using transcripts per million (TPM) values per gene (log2 transformed). Only high variance genes with adjusted p value smaller than 0.05 and present (mapped reads > 0) in more than 10 cells were selected.
  • WGCNA analysis To avoid gene modules due to co-expression within a single cell, we sub- sampled half of the cells 10 times, and calculated the topological overlap matrix for each sub- sampling. The final similarity matrix was taken as the minimum of all the topological overlap measure (TOM) similarity matrices. WGCNA clustering was performed using soft power of 4, cut height of 0.995, and minimum module size of seven. Clustering of cells was performed using hclust based on module eigengenes inferred by WGCNA and "ward" distance measure. Cells were grouped into four clusters.
  • TOM topological overlap measure
  • ChlP-Seq peaks associated with the given gene set show enrichment against the alternative gene set based on similar hypergeometric test at pvalue cutoff 0.05. Based on this test, Elk and SRF are selected as the top enriched ChlP-Seq peaks for vRG gene sets, and NRSF for oRG gene sets. To confirm whether the ChlP-Seq peaks are due to direct binding of the transcription factor, we confirmed the presence of known motifs for the factors within the ChlP-Seq peaks.
  • Cortical tissue sections were immersion fixed in 4% paraformaldehyde in PBS for 24 h at 4°C. Tissue was washed in PBS, transferred to 30% sucrose in PBS, stored in sucrose (Sigma) for 2 days (or until tissue sank). Tissue was embedded in Tissue-Tek O.C.T (Sakura Finetek,Torrance, CA) and stored at -80°C. For staining, 25 ⁇ coronal sections were cut, adhered to slides, and stored at -80°C. Slides were thawed and dried for 15 min at RT. PBS was dripped onto slide to wash away OCT and slides were then placed in 4% paraformaldehyde fixative for 20 min at RT.
  • Confocal images were acquired on a Leica TCS SP8 confocal microscope. Images were acquired using sequential acquisition of individual channels to prevent bleed through.
  • RGs radial glial progenitors and the complex intracellular molecular networks that generate them are essential for understanding human brain development.
  • RGs are present in the ventricular zone (VZ) and are bi-polar with an attached end foot at the ventricular surface ( Figure 5A) 1 .
  • outer subventricular zone (oSZ) RG cells (oRGs) can be distinguished by their position in the oSZ, a lack of an apical endfoot, and a basal process that can extend to the pial surface 2- " 4.
  • the oRGs are thought to be important in the cortical expansion observed in gyrified brains and the huge increase in size and complexity of the human brain.
  • Figure 5A 1 ' 5 .
  • RGs comprise only a small subset of cells in the human cortex
  • analysis of these cells has largely been limited to morphological descriptions with antibodies for only a couple markers to confirm cell identity 2- " 4 , molecular characterization of micro-dissected tissue which contains an unknown variety of cell types 6 ' 7 , or cells sorted based on cell surface markers 8 ' 9 ( Figure 5B). Because of these issues, we still lack specific molecular markers of oRG cells and have only a limited understanding of human progenitor diversity.
  • RG progenitors require transcriptomic profiles of larger numbers of single cells, ideally from targeted subpopulations.
  • Our strategy to study the diversity of progenitor cells in a developing human brain was to isolate individual progenitor cells from human fetal tissue based on the expression patterns of canonical transcription factors SOX2 and PAX6, profile the transcriptomes of single cells to identify the RG diversity, and computationally determine the gene expression networks controlling progenitor biology and lineage.
  • FIG. IE Live or fixed HI hESC RNA-Seq libraries were prepared transcriptomic profiling using the full length mRNA amplification method SmartSeq2 u .
  • the "Fixed" prep amplified comparable amounts of cDNA from individual fixed and live cells, and those cDNAs were prepared into RNA-Seq libraries and sequenced ( Figures 2A-2B and Figure 6). There were no significant differences in frequencies of reads mapping to different classes of RNA between live and fixed cells the "Fixed" prep ( Figure 2C and Figure 10A). Furthermore, Pearson correlation, and differential gene expression did not distinguish live from fixed cells ( Figures 2D and 2F).
  • Table 3 consists of differentially expressed genes in hESCs between conditions.
  • Differential expression is based on DESeq. Significantly differentially expressed genes (padj ⁇ 0.01) are shown with their fold change. The "LL LF DESeq genes” tab compares Live/Live verses Live/Fixed, and “LL LF DESeq genes” tab compares Live/Live verses Fixed/Fixed. Mitochondrial, Histone, rRNA genes are indicated, as well as genes previously shown to undergo differential polyadenylation 12. Fixed/Live comparisons were omitted since there were too many differentially expressed genes, and no differentially expressed genes were detected between Fixed/Live, and Fixed/Fixed at the indicated threshold.
  • the germinal zones of the human cortex including the VZ, inner, and outer
  • subventricular zones iSZ and oSZ
  • iSZ and oSZ have a mixture of progenitor and other cell types ( Figure 5A), making it difficult to resolve cell type-specific gene expression signatures by position alone 4 .
  • RGs in the VZ and oRGs in the oSZ have been reported to co-express PAX6 and SOX2, but lack the intermediate progenitor cell (IPC) marker TBR2 3 ' 4 .
  • IPC intermediate progenitor cell
  • Modules 1 and 2 reflected the division of the majority of SP from SPT cells, and contained canonical markers of RGs including VIM, and IPCs including EOMES (TBR2), HES6, and NEUROG1 (Figure 7E, Figure 15).
  • Modules 5 and 6 contained many cell cycle- related genes, and were enriched in SPT cells. Consistent with this, analysis of DNA content showed twice as many SPT cells are in the S-G 2 -M phases of the cell cycle as SP cells ( Figure 1 IB).
  • Module 3 revealed an RG subpopulation significantly enriched for the immediate early genes (IEGs) EGR1 and FOS 13.
  • SP cells expressing EGR1 and FOS still had some diversity based on the variable expression patterns of CXCL12, ANXA1 and CYR61 (data not shown).
  • FACS analysis confirmed FSC-Seq data that SP, but not the SPT cells could be divided into two subpopulations based on the expression of CYR61 protein ( Figure 16).
  • the module 4 eigengene was complementary to module 3 in the SP cells ( Figure 7E), and is composed of genes such as FAM107A, HOPX, and SLC01C1 ( Figure 7G). Many genes in module 4 have no previously known roles in cortical development, and FAM107A and ANXA1 are genes uniquely expressed in human but not mouse RG cells 15 .
  • Module 4 genes were enriched in the oSZ compartment relative to the VZ in 21 but not 15 or 16 PCW cortical samples based on analysis of the
  • HOPX + SOX2 + cells in the oSZ displayed oRG morphology with basally directed phospho- VIM + processes, and rarely exhibited apical process anchored at the ventricular surface (Figure 8D-8E).
  • HOPX + progenitors in the oSZ were proliferative since more than 15% of HOPX + SOX2 + cells co-stained with Ki67, while HOPX- SOX2+ cells (presumably IPCs) were significantly more proliferative (Figure 8F).
  • HOPX was a very specific marker of oRG progenitors: more than 80% of SOX2 + cells in the oSZ co- stained with HOPX, and less than 1% of HOPX + cells in the oSZ are SOX2 " ( Figure 8G-8H).
  • Coordination Center 18 was used to identify transcription factor occupancy at open sites. There was a statistically significant enrichment in SRF and ELK binding sites proximal to vRG genes, and NRSF/REST binding sites proximal to oRG specific genes ( Figure 81). It is known that SRF and ELK regulate FOS and EGRl 19 ' 20 , loss of SRF leads to higher expression of SOX2 in the mouse germinal zone 21 , and that HOPX inhibits the function of SRF in developing cardiac 22 ' 23. These findings point to a model in which SRF maintains expression of the vRG marker, and the activation of HOPX leads to the inhibition of SRF and the depletion of vRG genes.
  • HOPX expression is initiated as the oSZ begins to expand, it is first expressed in the VZ and the oSZ, and at later times is enriched in the oSZ ( Figure 17C).
  • the method we developed to study the diversity of human cortical progenitors is a new technology that enables targeted purification and efficient single-cell profiling of low frequency cell populations from primary tissue or other sources, obviating the need for transgenesis or viral labeling of specific cell types, and providing RNAseq data comparable to that obtained from live cells.
  • we used our transcriptional data to direct the computational analysis of existing epigenetic data and propose molecular network that controls the identity and lineage of vRGs and oRGs.
  • inventive embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed.
  • inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein.
  • a reference to "A and/or B", when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • the phrase "at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.
  • At least one of A and B can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

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Abstract

La présente invention concerne, selon certains aspects, des compositions, des procédés et des kits d'isolement d'ARN à partir d'échantillons biologiques fixés. Ces compositions, procédés et kits sont utiles pour isoler de l'ARN intact (c'est-à-dire ni dégradé, ni fragmenté), par exemple de l'ARN présentant un indice d'intégrité de l'ARN (RIN) ≥ 8,0.
PCT/US2016/025077 2015-03-30 2016-03-30 Procédés d'isolement d'arn de grande qualité à partir d'échantillons fixés WO2016161023A1 (fr)

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
CN111286542A (zh) * 2020-02-17 2020-06-16 浙江海洋大学 一种用于估测鱼类死亡时间的引物及其应用
US11168323B2 (en) 2017-06-01 2021-11-09 Nantomics Llc DNA stabilization of RNA
WO2022188054A1 (fr) * 2021-03-10 2022-09-15 Nanjing University Procédés et réactifs pour le multiplexage d'échantillons pour le séquençage d'arn monocellulaire à haut débit

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US20070026432A1 (en) * 2005-05-31 2007-02-01 Invitrogen Corporation Separation and purification of nucleic acid from paraffin-containing samples
US20130280787A1 (en) * 2010-06-14 2013-10-24 Qiagen Gmbh Extraction of nucleic acids from wax-embedded samples
WO2014052551A1 (fr) * 2012-09-28 2014-04-03 Cepheid Procédés d'extraction d'adn et d'arn à partir d'échantillons tissulaires fixés incorporés dans de la paraffine

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US20070026432A1 (en) * 2005-05-31 2007-02-01 Invitrogen Corporation Separation and purification of nucleic acid from paraffin-containing samples
US20130280787A1 (en) * 2010-06-14 2013-10-24 Qiagen Gmbh Extraction of nucleic acids from wax-embedded samples
WO2014052551A1 (fr) * 2012-09-28 2014-04-03 Cepheid Procédés d'extraction d'adn et d'arn à partir d'échantillons tissulaires fixés incorporés dans de la paraffine

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11168323B2 (en) 2017-06-01 2021-11-09 Nantomics Llc DNA stabilization of RNA
US11981893B2 (en) 2017-06-01 2024-05-14 Nantomics Llc DNA stabilization of RNA
CN111286542A (zh) * 2020-02-17 2020-06-16 浙江海洋大学 一种用于估测鱼类死亡时间的引物及其应用
CN111286542B (zh) * 2020-02-17 2023-05-16 浙江海洋大学 一种用于估测鱼类死亡时间的引物及其应用
WO2022188054A1 (fr) * 2021-03-10 2022-09-15 Nanjing University Procédés et réactifs pour le multiplexage d'échantillons pour le séquençage d'arn monocellulaire à haut débit

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