cDNA Synthesis for Real Time
(Using TaqMan Reverse Transcription Reagents)
1. Combine the following in a 0.6ml eppendorf tube:
+RT reaction
RNA (1µg)
10x TaqMan RT Buffer
25mM MgCl2
dNTPs
50µM Random Hexamers
RNase Inhibitor (20U/µl)
Multiscribe RT (50U/µl)
Total
1rxn (µl)
up to 38.5
10
22
20
5
2
2.5
100µl
-RT reaction
RNA (1µg)
10x TaqMan RT Buffer
25mM MgCl2
dNTPs
50µM Random Hexamers
RNase Inhibitor (20U/µl)
Total
1rxn (µl)
up to 41
10
22
20
5
2
100 µl
2. Incubate in 9700:
25ºC 10min
48 ºC 30min
95 ºC 5min
3. According to Applied Biosystems, you can assume that the amount you put into
the reaction is the amount you get back. So, 1µg of RNA leads to 1µg of cDNA.
From here, you can make your dilutions for real time experiments.