Bio-Rad SingleShot™ Cell Lysis RT-qPCR Kits - Manual (Page 10)

© 2014 Bio-Rad Laboratories, Inc. 10042474
SingleShot ™ SYBR ® Green Kit
Table 8. Setup of reverse transcription reactions to optimize input 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
* Includes 5x iScript advanced reaction mix.
* Scale all components proportionally according to sample number and reaction volumes.
Table 9. Preparation of qPCR reaction mix to determine optimal input lysate amount.*
Component Volume per 20 µl Reactions, µl Final Concentration
SsoAdvanced ™ Universal SYBR ®
Green Supermix (2x)
10 1x
SingleShot ™ SYBR ® Green qPCR
Control Assay
1 1x
cDNA (add at step 7) 7 —
Nuclease-Free Water Variable —
Total reaction mix volume 20 —
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.
Using the SingleShot RNA Control Assay to Determine Optimal Input Lysate Volume
1. Resuspend the RNA control template in 200 µl of nuclease-free TE buffer pH 7.5.
2. Prepare cell lysate from either adherent (see Processing of Adherent Cells in a
96-Well Culture Plate section) or suspension cells (see Processing of Nonadherent
Cells in a 96-Well PCR Plate section) with an optimal number of input cells.
3. Vary input cell lysate in the reverse transcription reactions as shown in Table 8.
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.