Anyone can guide me through from scratch how to do gene expression for microRNAs by real time PCR. I mean how specific primers are designed for miRs. If normal reverse-transcribed cDNA will also work for miRs or need extra step. What kind of program is used in the thermocycler. Need teeny N tiny details, please
Thanks, Markus,
This will definitely help me. I appreciate you very much.
Best regards, Subhash
Thanks a lot, Alia,
I appreciate your valuable information. Once I will start doing it, I will ask you more questions if I will have.
Best regards,
Subhash
To asses mature microRNA expression in Dendritic Cells, we used two-step qRT-PCR, with TaqMan microRNA assays (Applied Biosystems). For microRNA cDNA synthesis, a 15 μL reaction volume, composed of 1.5 μL of Buffer (10x), 0.15 μL 100 mM dNTPs (100 mM), 1.0 μL reverse transcriptase, 0.19 μL RNAse inhibitor (20 U/μL), and 3.0 μL of each specific microRNA primer, was mixed with 10 ng of total RNA. RT reaction is incubated 30 minutes of 16°C, 30 minutes of 42°C, and 5 minutes of 85°C. Duplicate real-time PCR reactions are performed in a Roche LigthCycler 2.0. Reaction mix was composed of 1x LigthCycler TaqMan Master (Roche), 1X of each specific microRNA probe, and 2.5 uL of specific microRNA cDNA (diluted 1 : 3). These were followed by 10 minutes 95°C for preincubation, 45 cycles of amplification program consisting of 95°C 10 sec, 60°C 40 sec, and 72°C 5 sec (fluorescence acquisition). To asses possible bias for reference gene selection RNU6B, U6 and let7a were used as reference genes, and relative quantification was calculated by the formula 2-(Cp microRNA target—Cp U6 or let7a). All microRNA assays were tested for reproducibility and linearity (PCR efficiency was between 1.9 and 2.0 for all assays).
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