Protocol: cDNA Synthesis and qRT-PCR using the BioRad MyiQ
Adam Skibinski, Kuperwasser Lab, 3-9-11
Reagents needed
RNA samples
iScript 5x Reaction Buffer (blue tubes, -20°C common freezer)
iScript Reverse Transcriptase (yellow tubes, -20°C common freezer)
SYBR Green Supermix (orange tubes, -20°C common freezer)
RNase-free water
RT-PCR primers (2.5 µM)
Axygen PCR plates (Drawer by PCR machine)
Bio-Rad sealing tape (Drawer by PCR machine)
Procedure
1. Thaw RNA samples, spec using NanoDrop (8th floor Stearns). Thaw iScript 5x reaction buffer.
2. For each sample, set up the following reaction in a 0.2 ml PCR tube:
| 1 µg RNA sample | varies |
| 5x iScript buffer | 4 µl |
| iScript reverse transcriptase | 1 µl |
| RNase-free water | to 20 µl |
3. Mix tubes by vortexing and spin down
4. Place tubes in PCR machine. Turn on the machine, select ‘Registered User’ and select Charlotte. Go to the “protocols” menu and select ‘cdnasynthesis.’ Set the reaction volume to 20 µl.
cdnasynthesis
25°C for 5 min
42°C for 30 min
85°C for 5 min
4°C hold
5. After cDNA synthesis is complete, dilute samples to 100 µl each by adding 80 µl of RNase-free water. Samples are stable at -20°C or 4°C for a few weeks.
