cDNA Synthesis and qPCR

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