Bio-Rad SingleShot™ Cell Lysis RT-qPCR Kits - Manual (Page 13)

© 2014 Bio-Rad Laboratories, Inc. 10042474
SingleShot ™ SYBR ® Green Kit
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 species or culture conditions,
the 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 with PBS to remove contaminants in the
culture medium
■ ■■■ 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 treating
the cells
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 by replacing reagents one
by one; use new aliquots of assays
■ ■■■ Evaluate the assay design for primer dimer formation;
perform gradient PCR to optimize the annealing
temperature; use a primer matrix to determine the
optimal primer concentration