Bio-Rad SingleShot™ Cell Lysis RT-qPCR Kits - Manual (Page 9)

© 2014 Bio-Rad Laboratories, Inc. 10042473
SingleShot ™ Probes Kit
4. 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.
5. Set up qPCR reactions following instructions in Table 9. Do not add cDNA
until step 7.
6. Mix the qPCR reaction mix thoroughly to ensure homogeneity and dispense equal
aliquots into each PCR tube or into the wells of a PCR plate. Use good pipetting
technique to ensure assay precision and accuracy.
7. Add cDNA to the PCR tubes or wells containing qPCR reaction mix (prepared using
Table 9), seal tubes or wells with flat caps or optically transparent film, and gently
vortex to ensure thorough mixing of the reaction components. Spin the tubes
or plate to remove any air bubbles and to collect the reaction mixture in the
vessel bottom.
8. Program the thermal cycling protocol on a real-time PCR instrument according
to Table 5.
* Includes 5x iScript advanced reaction mix, iScript advanced reverse transcriptase, and nuclease-free H
2
O.
Table 8. Setup of reverse transcription reaction to optimize input cell lysate amount.
Input Lysate, % Lysate Volume, µl
RNA Control
Template, μl
2x RT
Master Mix, μl
Nuclease-Free
H
2
O, μl
10 2 1 10 7
20 4 1 10 5
30 6 1 10 3
40 8 1 10 1
45 9 1 10 0
* Scale all components proportionally according to sample number and reaction volumes.
Table 9. Preparation of qPCR reaction mix for RNA control assay.*
Component
Volume per 10 µl
Reaction, µl
Volume per 20 µl
Reactions, µl
Final
Concentration
SsoAdvanced Universal Probes
Supermix (2x)
5 10 1x
SingleShot ™ Probes qPCR
Control Assay
0.5 1 1x
cDNA (add at step 7) 1–2 2–4 —
Nuclease-Free Water Variable Variable —
Total reaction mix volume 10 20 —