cDNA synthesis
Follow protocol from Biorad iScript reverse transcriptase (described below).
In 8-strip PCR tubes, mix the following reagents for each reaction:
(include controls with no RT)
5x iSript reaction buffer 4 μl
RNA 1 μg total
Reverse transcriptase 1 μl
RNAse-free water (volume to 20 μl total)
In PCR machine, run “cDNA” program
25ºC, 5 min
42ºC, 30 min
85ºC, 5 min
4ºC, 10min
Store cDNA at -20ºC.
qPCR
Set up master mixes for 10 µL reactions as follows:
(For total amount of master mix, multiply each by total number of reactions plus two. Perform each reaction in triplicate).
Mix#1:
2x SYBR Green mix 5 µL
20 µM forward gene-specific primer 0.25 µL
20 µM reverse gene-specific primer 0.25 µL
Mix#2
cDNA 0.5 µL
Water 4 µL
Combine reactions in 384 well plate, adding Mix#1 to wells first, followed by Mix#2.
In qPCR machine, run 2-step protocol with melt curve
95ºC for 3 min
Repeat for 40 cycles:
95ºC for 10 sec
55ºC for 30 sec
Melt curve:
55-95ºC, 0.5ºC increments