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.
