Bio-Rad SingleShot™ Cell Lysis RT-qPCR Kits - Manual (Page 11)

© 2014 Bio-Rad Laboratories, Inc. 10042473
SingleShot ™ Probes Kit
Appendix A
Table 1. Setting up SingleShot cell lysis reactions.
* Prepare excess SingleShot cell lysis master mix to ensure enough reagent is available.
Step Guidelines
Preparing the
cell culture
Number of wells 384 96 48 24 12 6
Cell numbers per
well at harvest 10–
5 x 10 4
10–
1 x 10 5
10–
2 x 10 5
10–
4 x 10 5
10–
8 x 10 5
10–
1 x 10 6
Washing the
cells with PBS Volume of PBS
per well, µl
30 125 250 500 1,000 2,000
Preparing the
SingleShot
cell lysis
master mix
SingleShot Cell
Lysis Buffer, µl
12 48 96 192 384 768
Proteinase K, µl 0.25 1 2 4 8 16
DNase, µl 0.25 1 2 4 8 16
Total volume
per well, µl
12.5 50 100 200 400 800
Troubleshooting Guide
Problem Potential Cause Solution
No amplification
in the RT-qPCR
reaction
Delayed Cq
values seen in
RNA detection
■
■■■ Cell lines may contain high
levels of PCR inhibitors
■
■■■ Excess number of cells
used in the lysis reaction
■ ■■■ Excess cell culture
medium carryover
■
■■■ Excess lysate used in
the RT-qPCR reaction
■
■■■ Depending on the cell type or culture conditions, the
input number of cells or percentage lysate may require
optimization (see Optimizing Input Cell Number and
Input Lysate Amount section)
■
■■■ Generally ≤10 5 cells can be used successfully in
the SingleShot procedure, but if RT or qPCR fails,
try using 5- to 10-fold fewer cells
■ ■■■ Wash cells twice with PBS to minimize inhibition from
excess cell culture medium carryover
■
■■■ Remove as much of the culture medium and PBS
as possible
■ ■■■ Use a freshly prepared SingleShot cell lysis master
mix; keep on ice and use within 2 hr
■ ■■■ Make sure DNase and proteinase K are added in the
SingleShot cell lysis master mix before cell lysis
Genomic DNA
is amplified as
seen in the
no-RT control
■ ■■■ Incomplete gDNA
digestion
■
■■■ DNase and Proteinase K
were not added to the
lysis reaction
■ ■■■ Repeat the lysis step. Ensure DNase is added,
the thermal cycling conditions are correct,
and the thermal cycler is working properly
Signal in no
template control
(NTC) reaction
■
■■■ DNA contamination (NTC
melt peak T
m
is identical
to the target gene melt
peak T
m
)
■
■■■ Primer dimers (NTC melt
peak is broad with a T
m
~65–75°C)
■
■■■ Examine the workflow to identify potential
contamination sources; replace reagents one by one
until the contamination source is identified. Be sure to
use filtered pipet tips
■
■■■ Evaluate the assay design for primer dimer formation;
use gradient PCR to optimize the annealing
temperature; use a primer matrix to determine
the optimal primer concentration.