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Single cell qPCR protocol. 1. Sort individual cells into 4ul Rnase free water. a. Use low profile pcr tube strips for use with the CFX96 b. Use the Biorad adhesive sealant and separate individual strips with a razor blade i. If you sort more than eight cells, you will want to keep the strips separate for processing purposes. Also allows you to trouble shoot protocols one strip at a time (aka 8 cells at time). c. Immediately freeze on dry ice at the cell sorter d. Store in the -80C 2. Cell lysis a. Take tube strips from -80C and transport to the CFX96 on ice b. Use the cell-melt program (95C for 10 minutes) c. Once complete, immediate put strips on ice and remove adhesive seal while it is still hot. d. Quickly add RT mix, then primer mix (see step 3) 3. Reverse Transcription a. RT reactions consist of 7ul RT reaction mix:4ul cell lysis:4ul Primer mix. (15ul total) b. Use 1ul of 1uM RT primer for miRNA or siRNA and 1ul of 1uM poly d(T) primer for mRNA (make a master mix and aliquot it out 1ul RT primer:1 ul poly d(T) primer: 2ul water) c. RT master mix (ABI), directly scale up mix (aka multiply all components by 8 if you are doing 8 RT reactions). Component Amount (ul) Buffer 1.65 NTP 0.125 RT 1.1 Inhibitor 0.21 Water 4.615 Total 7.7 4. Rnase H treatment a. Add 0.5ul of RNase H (NEB) to each RT reaction and incubate at 37C for 20 minutes 5. Preamplification a. Make a master mix using the following (directly scale up as before) b. Each species you want to detect requires its own preamp reaction (aka one for a miRNA and a separate one for a single mRNA) c. It may be possible to preamplify all mRNA to be detected in a single reaction, but that will requires controls to insure that there is no primer-dimer interactions between primer sets d. Do preamps on the CFX96 e. Use the Amplitaq Gold master mix (ABI) for miRNA preamps f. Use the SyberGreen master mix (Biorad) for mRNA preamps Component Amount (ul) Buffer/enzyme mix 15.5 Primer F (10 uM) 0.6 Primer R (10 uM) 0.6 Rnase treated RT reaction 2 Water 12.3 Total 31 6. Dilute Preamplification reactions a. Transfer preamp reaction to a 1.5ml eppitube b. Rinse the tube strip well that contained the preamp reaction with 90ul water and use this “wash” to dilute the preamp reaction to a total of 120ul. c. Samples can be frozen at -20C or directly used for qPCR analysis 7. qPCR analysis a. Make separate master mixes for each species to be detected b. Directly scale all mixtures as described above c. Always use the same reaction master mix for both the preamp and qPCR reactions d. Use the Amplitaq Gold master mix (ABI) for miRNA preamps e. Use the SyberGreen master mix (Biorad) for mRNA preamps f. qPCR for miRNA, siRNA, and mRNA can be done on the same 96 well plate g. Detect each species in triplicate h. miRNA mix: Component Amount (ul) Buffer/enzyme mix 10.5 Primer F (10 uM) 0.4 Primer R (10 uM) 0.4 Taqman probe (1uM) 0.4 Preamp reaction 0.5 Water 8.8 Total 21 i. mRNA mix Component Amount (ul) Buffer/enzyme mix 10.5 Primer F (10 uM) 0.4 Primer R (10 uM) 0.4 Preamp reaction 0.5 Water 8.8 Total 21 j. Cycling parameters i. 95.0 C for 10:00 ii. 95.0 C for 0:30 iii. 58.9 C for 0:30 (optimized for Citrine/mCherry/mir124) iv. + Plate Read v. GOTO 2, 50 more times vi. 40.0 C for 1:00 vii. Melt Curve 40.0 to 95.0 C, increment 0.5 C, viii. 0:05 + Plate Read ix. END 8. Notes a. Be sure to construct calibration curves and analyze them using the same cell handling protocol as described above. Do, at a minimum, a five point calibration curve. Each point of the curve should be a 10x dilution. Be sure that your efficiency is between 90- 100% and that your slope is as close to 1.5 as possible. Annealing temperatures will need to be optimized in order to meet these criteria. b. qPCR Calibration curves of preamplified standards will parallel a qPCR calibration curves of RT reactions. c. Use an exponential fit to calculate calibration curves. Convert Ct values to molecules and be sure to normalize the values by the weight of a single molecule of each species being detected. This will allow the calculation of how many molecules were present in each cell measured.
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