Bio-Rad SingleShot™ Cell Lysis RT-qPCR Kits - Manual (Page 4)

© 2014 Bio-Rad Laboratories, Inc. 10042474
SingleShot ™ SYBR ® Green Kit
Table 3. Preparation of reverse transcription reaction.
Component Volume per 20 μl Reaction, µl
5x iScript Advanced Reaction Mix 4
iScript Advanced Reverse Transcriptase 1
Cell Lysate 4–9
Nuclease-Free Water Variable
Total reaction mix volume 20
2. Incubate the complete reaction mix in a thermal cycler using the following protocol:
reverse transcription for 30 min at 42°C followed by RT inactivation for 5 min
at 85°C.
3. Proceed to the Preparation of qPCR Reactions section, or store the cDNA
at –20°C.
Recommendations for the use of no-RT control
■ ■
Interference of gene expression analysis by gDNA carryover in cell lysate
samples can be tested by setting up a no-RT control reaction
■ ■
The reverse transcriptase volume in a no-RT control reaction should be replaced
with water
■ ■
The same amount of cell lysate used in the RT reaction should be used in the
no-RT reaction to ensure similar carryover of cDNA synthesis components in a
qPCR reaction
Preparation of Reverse Transcription Reactions
1. For optimal results, reactions should be assembled on ice. Prepare the reverse
transcription reaction according to the directions in Table 3. Mix thoroughly by
pipetting up and down several times.