Name: Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR (Video) | JoVE Journal
Uploaded: 2014-05-29T13:00:00.000+00:00
Duration: 9 min 3 s
Description: Learn single-cell quantitative RT-PCR to profile gene expression in individual human embryonic stem cells. This method reveals population heterogeneities using fluorescent activated cell sorting and amplification techniques.

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5 minutes

We would like to thank members of the Lee lab for valuable discussions on the manuscript. Work in the Lee lab was supported by grants from Robertson Investigator Award of New York Stem Cell Foundation and from Maryland Stem Cell Research Fund (TEDCO).

1. Preparation of a 96-well Plate
<ol>
	<li>Mix 1 &#956;l of Single Cell DNase1 to 9 &#956;l Single Cell Lysis Solution.</li>
	<li>Put the 10 &#956;l mixed solution in each well of 96-well PCR plate.</li>
</ol>
2. Detaching hESCs for FACS Purification
<ol>
	<li>Detach OCT4::EGFP ES cell line from the 60 mm dish with 1 ml Accutase for 20 min, at 37 &#176;C, which were neutralized with human ES media.</li>
	<li>Prepare cell population in 1 ml FACS buffer and adjust the cell to 1 x 106 cells/ml.</li>
	<li>Pass the cell sample through a 35 &#956;m cell strainer cap tube.</li>
	<li>Store the tube in ice before cell sorting.</li>
</ol>
3. Lysis of FACS-purified Single Cell in Each Well of the 96-well Plate
<ol>
	<li>Sort the sample for EGFP positive cells on a cell sorter with a trained operator. Put the single cell into Single Cell Lysis/DNase1 solution in 96-well PCR plate. If necessary, the 96-well plate with sample can be stored in a -80 &#176;C deep freezer less than one month.</li>
	<li>Incubate samples 5 min at RT for cell lysis.</li>
	<li>Add 1 &#956;l of Stop Solution to stop lysis reaction.</li>
	<li>Incubate 2 min at RT.</li>
</ol>
4. Reverse Transcription
<ol>
	<li>Add to each a 0.5 &#956;l aliquot of 20 &#956;M SMA-T15, SMA-A.</li>
	<li>Add 4 &#956;l 5X buffer, 2 &#956;l DTT, 1 &#956;l Reverse Transcriptase, and 1 &#956;l dNTP to each well.</li>
	<li>Perform reverse transcription in a thermal cycler.
	<ol>
		<li>Set the thermal program at 42 &#176;C &#215; 90 min and inactivate Reverse Transcriptase at 85 &#176;C &#215; 5 min.</li>
	</ol>
	</li>
</ol>
5. Amplification
<ol>
	<li>Add 4 &#956;l of ExoSAP-IT reagent to each reverse transcribed sample.
	<ol>
		<li>Incubate samples at 37 &#176;C for 15 min and 80 &#176;C for 15 min to inactivate the ExoSAP-IT reagent.</li>
	</ol>
	</li>
	<li>Prepare PCR reaction mix with SMA-p2 (2 nM)</li>
	<li>Add 10 &#956;l of PCR reaction mix to each reverse transcribed sample.</li>
	<li>Perform the amplification, consisting of 20 cycles of denaturation (94 &#176;C for 30 sec), annealing (57 &#176;C for 30 sec), and extension (68 &#176;C for 10 min).</li>
</ol>
6. qRT-PCR Performance
<ol>
	<li>Add 10 &#956;l of 2X SYBR Green PCR Master Mix, 1 &#956;l amplified cDNA, 2 nM primers, and 7 &#956;l water to each well.
	<ol>
		<li>Set the program followed by 95 &#176;C for 3 sec, 60 &#176;C for 30 sec x 40 cycles.</li>
	</ol>
	</li>
	<li>Perform in duplicate for technical errors.</li>
</ol>

<ol><li>Miyanari, Y., Torres-Padilla, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Control+of+ground-state+pluripotency+by+allelic+regulation+of+Nanog%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Control of ground-state pluripotency by allelic regulation of Nanog.</a> Nature. 483, 470-473 (2012).</li>
<li>Leitch, H. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Embryonic+germ+cells+from+mice+and+rats+exhibit+properties+consistent+with+a+generic+pluripotent+ground+state%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Embryonic germ cells from mice and rats exhibit properties consistent with a generic pluripotent ground state.</a> Development. 137, 2279-2287 (2010).</li>
<li>Ramskold, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Full-length+mRNA-Seq+from+single-cell+levels+of+RNA+and+individual+circulating+tumor+cells%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells.</a> Nat Biotechnol. 30, 777-782 (2012).</li>
<li>Stewart, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clonal+isolation+of+hESCs+reveals+heterogeneity+within+the+pluripotent+stem+cell+compartment%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Clonal isolation of hESCs reveals heterogeneity within the pluripotent stem cell compartment.</a> Nat Methods. 3, 807-815 (2006).</li>
<li>O'Malley, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-resolution+analysis+with+novel+cell-surface+markers+identifies+routes+to+iPS+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">High-resolution analysis with novel cell-surface markers identifies routes to iPS cells.</a> Nature. 499, 88-91 (2013).</li>
<li>Nichols, J., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Naive+and+primed+pluripotent+states%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Naive and primed pluripotent states.</a> Cell Stem Cell. 4, 487-492 (2009).</li>
<li>Kim, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((miR-371-3+expression+predicts+neural+differentiation+propensity+in+human+pluripotent+stem+cells%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">miR-371-3 expression predicts neural differentiation propensity in human pluripotent stem cells.</a> Cell Stem Cell. 8, 695-706 (2011).</li>
<li>Mortazavi, A., Williams, B. A., Mccue, K., Schaeffer, L., Wold, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mapping+and+quantifying+mammalian+transcriptomes+by+RNA-Seq%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Mapping and quantifying mammalian transcriptomes by RNA-Seq.</a> Nat Methods. 5, 621-628 (2008).</li>
<li>Pan, Q., Shai, O., Lee, L. J., Frey, J., Blencowe, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deep+surveying+of+alternative+splicing+complexity+in+the+human+transcriptome+by+high-throughput+sequencing%5BTitle%5D)+AND+%22Nat+Genet%22%5BJournal%5D)">Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing.</a> Nat Genet. 40, 1413-1415 (2008).</li>
<li>Wang, E. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alternative+isoform+regulation+in+human+tissue+transcriptomes%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Alternative isoform regulation in human tissue transcriptomes.</a> Nature. 456, 470-476 (2008).</li>
<li>Hockemeyer, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+engineering+of+human+pluripotent+cells+using+TALE+nucleases%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">Genetic engineering of human pluripotent cells using TALE nucleases.</a> Nat Biotechnol. 29, 731-734 (2011).</li>
<li>Lee, G., Chambers, S. M., Tomishima, M. J., Studer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Derivation+of+neural+crest+cells+from+human+pluripotent+stem+cells%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Derivation of neural crest cells from human pluripotent stem cells.</a> Nat Protoc. 5, 688-701 (2010).</li>
<li>Zhu, Y. Y., Machleder, E. M., Chenchik, A., Li, R., Siebert, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reverse+transcriptase+template+switching%3A+A+SMART+%28TM%29+approach+for+full-length+cDNA+library+construction%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">Reverse transcriptase template switching: A SMART (TM) approach for full-length cDNA library construction.</a> Biotechniques. 30, 892-897 (2001).</li>
<li>Pan, X. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two+methods+for+full-length+RNA+sequencing+for+low+quantities+of+cells+and+single+cells%5BTitle%5D)+AND+%22P+Natl+Acad+Sci+USA%22%5BJournal%5D)">Two methods for full-length RNA sequencing for low quantities of cells and single cells.</a> P Natl Acad Sci USA. 110, 594-599 (2013).</li>
<li>Tang, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA-Seq+analysis+to+capture+the+transcriptome+landscape+of+a+single+cell%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">RNA-Seq analysis to capture the transcriptome landscape of a single cell.</a> Nat Protoc. 5, 516-535 (2010).</li>
</ol>

The authors have nothing to disclose.

Single cell gene profiling could be a major tool to predict functionality of a single cell or an entire population. Due to technical limitation, whole gene profiling analysis has been restricted to population averages. Variations in gene expression patterns and levels between individual cells and the subpopulations have been proposed to cause erroneous interpretation. Such diverse cellular aspects can be found in hESCs and their heterogeneity causes subtly different ability for maintaining pluripotency and fate specifica...

Most higher eukaryote populations are heterogeneous thus with analysis of pooled population, it is often difficult to interpret their cellular features. Individual cells within a population may be subtly different, and these differences can have important consequences for the property and function of the entire population1,2. Especially, human embryonic stem cells (hESCs) are known to be heterogeneous, which causes different levels of pluripotency and diverse potentials to lineage specification in delicately distinctive ways3,4. For example, different cell surface antigens can be used to categorize undifferentiated pluripotent stem cells,5 and the Austin Smith group proposed different levels of pluripotency in mouse embryonic stem cells, based on their morphology, differentiation propensity and dependency of signaling pathway6. This phenomenon was hypothesized in human embryonic stem cells7. Whereas the overall studies were performed among different stem cell lines, not individual single stem cells, it could be very intriguing to analyze different levels of pluripotency at the single cell level, which potentially affects their differentiation capacities toward all somatic cell lineages.
Cellular and molecular heterogeneity could be dictated by transcription profiling, which is called the &#8216;single cell transcriptome&#8217; and emphasizes new approaches for quantifying gene expression levels8-10. For analysis of gene expression levels in individual cells, we developed a simple, but robust protocol of single cell quantitative RT-PCR. We confirmed the efficacy and feasibility of our protocol by comparing each half of single cell lysates as well as serially diluted total RNAs of hESCs, resulting in minimal technical variations and differences. Further, we used a genetic reporter line to isolate homogenous population of hESCs using gene targeting system. &#160;The donor vector for targeting OCT4 locus (OCT4-2A-EGFP-PGK-Puro construct) and a pair of TALEN plasmids were used11. The donor vector and a pair of TALEN plasmids were introduced into hESCs (H9, WA09) using our nucleofection and clonal selection protocol and maintenance of hESCs was performed based on our routine protocol12. We confirmed this genetic reporter line express EGFP for OCT4 expression in OCT4::EGFP hESCs.
Our result demonstrates that individual hESCs (sorted by OCT4::EGFP strongly positive cells) hold high levels of OCT4 expression, but different levels of NANOG expression. So, our single cell gene expression assay should be useful to study population heterogeneities of pluripotent stem cells.

<table><tbody><tr><td>96-well PCR Plate</td><td>USA scientific</td><td>1402-8900</td><td></td></tr><tr><td>Ambion Cell Lysis Kit</td><td>Life Technologies</td><td>4458235</td><td></td></tr><tr><td>SMARTScribe Reverse Transcriptase</td><td>Clonetech</td><td>639536</td><td></td></tr><tr><td>ExoSAP-IT</td><td>USB</td><td>78200</td><td></td></tr><tr><td>Platinum Taq DNA polymerase High Fidelity</td><td>Invitrogen</td><td>11304</td><td></td></tr><tr><td>10 mM dNTP Mix, PCR Grade</td><td>Invitrogen</td><td>18427</td><td></td></tr><tr><td>SYBR Universal 2X Master Mix</td><td>Kapa biosystem</td><td>KR0389</td><td></td></tr><tr><td>Accutase</td><td>Innovative Cell Tech</td><td>S-1100-1</td><td></td></tr><tr><td>FACS buffer</td><td></td><td></td><td>45 ml PBS, 5 ml a-MEM, 100 &amp;mu;l DNase, filter sterilized</td></tr><tr><td>35 &amp;mu;m cell strainer cap tubes</td><td>BD Biosciences</td><td>352235</td><td></td></tr></tbody></table>

profiling individual human embryonic stem cells by quantitative rt-pcr

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Efficient and robust single cell RNA amplification
To minimize the transcriptional variation among hESCs, we used OCT4::EGFP hESC clone for FACS purification. After sorting OCT4::EGFP positive cells into a 96-well plate, each cell is lysed in lysis buffer and converted poly(A)+ RNA to full length cDNA using SMA-T15 (GACATGTATCCGGATGTTTTTTTTTTTTTTTT) primer and anchoring with SMA-A (ACATGTATCCGGATGTGGG) by using SMART template switching technology. The excess oligonuc...

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

The overall goal of this procedure is to profile gene expression levels of individual human embryonic stem cells using single cell quantitative reverse transcription polymerase chain reaction, or R-T-P-C-R. This is accomplished by first using fluorescent activated cell sorting to sort individual cells into each well of a 96 well plate containing cell lysis buffer. The second step is to reverse transcribe RNA to generate CDNA from single cell lysates.Next, the CDNA is amplified by polymerase chain reaction. The final step is to perform quantitative R-T-P-C-R on the amplified CDNA. Ultimately, this assay is useful for studying gene expression in heterogeneous populations such as human embryonic stem cells.The main advantage of this technique over existing single cell quantitative artificial vessel is that this method, EEG and more robust Prior to fax purification. The OCT four EGFP human embryonic stem cells or Hess are detached from the 60 millimeter dish. Remove all medium, add one milliliter of Accutane and incubate at 37 degrees Celsius for 20 minutes.Neutralize Accutane with one milliliter of human ES media centrifuge to pellet the cells after discarding the supernatant resuspend cells in fax buffer and adjust the concentration to one times 10 to the six cells per milliliter. Next, pass the cell sample through a 35 micrometer cell strainer cap tube. Store the tube on ice before cell sorting.Begin this procedure by preparing a master mix containing one microliter of single cell D Ns one and nine microliters of single cell lysis solution. For each, well transfer 10 microliters of the mix solution to each well of a 96 well PCR plate for fax analysis program, the cell sorter to sort single EGFP positive cells into the wells of the 96 well PCR plate after cell sorting, incubate the samples for five minutes at room temperature for cell lysis. To stop the lysis reaction, add one microliter of stop solution and incubate at room temperature for two minutes.To begin this procedure first add to each well of the 96 well plate a 0.5 microliter Eloqua of 20 micromolar SMAT 15 and SMAA primers. Heat the samples at 80 degrees Celsius for five minutes. Then add four microliters of five x buffer, two microliters of DTT, one microliter of reverse transcriptase, and one microliter of D and tps.To each well perform reverse transcription in a thermal cycler. Set the thermal program at 42 degrees Celsius for 90 minutes, followed by 85 degrees Celsius for five minutes. To inactivate the reverse transcriptase when reverse transcription is complete, add four microliters of exo reagent to each CDNA sample incubate at 37 degrees Celsius for 15 minutes to digest excess oligonucleotides glides, followed by 80 degrees Celsius for 15 minutes.To inactivate the exo reagent, prepare the PCR reaction mix with two nano molar of the SMAP two primer. Add 10 microliters of the PCR reaction mix. To each reverse transcribed sample.Perform the amplification consisting of 20 cycles of denaturation, a kneeling and extension after amplification of the samples performed two sets of quantitative R-T-P-C-R reactions in separate plates for confirming the R-T-P-C-R results to each well of the 2 96 well plates add 10 microliters of two x cyber green. PCR master mix one microliter of amplified CDNA two primers and seven microliters of water. Set the program for 95 degrees Celsius for three seconds, followed by 60 degrees Celsius for 30 seconds.For 40 cycles, the a kneeling step is removed to reduce the error of primer. A kneeling in this study, microgram amounts of total RNA of Hess were diluted down to nano and picogram levels to assess technical variability and detection of difference on low amounts of total RNA quantitative R-T-P-C-R of the G-A-P-D-H gene was performed using MR.NA of pooled husks serially diluted from 10 nanograms per microliter to 0.1 picograms per microliter. Each dot shows the CT value of serially diluted mRNA and single cell mRNA sorted by fax.A correlation was observed between each sample and QR TPCR results with several single cells showing similar CT values. Fax was used to sort and select strong EEG FP positive cells from an OCT four EGFP reporter HESC line. The results show an EGFP positive population of approximately 60%compared to the negative population to validate consistency among different batches of reverse transcription and amplification reactions.Fax purified single cell lysates were divided into half and the protocol applied to each half for batch comparisons. A significant correlation between the OCT four CT value of each half of single cell lysates was observed. A heat map presentation of single husk gene expression shows a consistent and robust level of OCT four expression, but varying levels of another stem cell marker gene, NAN og taken together our results demonstrate that this single cell gene expression assay is useful for studying population heterogeneities of pluripotent stem cells.After watching this video, you should have a good understanding of how to perform the QITP for single cell transcriptome properly.

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Learn single-cell quantitative RT-PCR to profile gene expression in individual human embryonic stem cells. This method reveals population heterogeneities using fluorescent activated cell sorting and amplification techniques.

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

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