Table 1. Lysis Buffer Composition
Component
Volume per sample (µl)
Nuclease Free Water
5x ImProm-II Buffer
15
10
10% Triton X-100/Nonidet P40
8
dNTPs (10 mM)
MgCl2 (25 mM)
RNase-Free DNase
2.5
2.5
2
Total Volume
40
Comment
The final RT volume will
eventually be 50 µl (see below)
The final concentration of Triton
X-100 and Nonidet P40 is 2%
each. The enzymatic activities of
DNase and reverse transcriptase
are not affected under these
conditions.
Table 2. RT Master Mix
Component
Volume per sample (µl)
Nuclease Free Water
ImProm-II Reverse
Transcriptase
RT Primers (1 µM each)
2.5
2.5
Comment
5
The final concentration of each RT
primer is 100 nM.
Total Volume
10
The total volume for RT is 50 µl.
Table 3. qPCR Master Mix
Component
Volume per sample (µl)
Nuclease Free Water
2x SsoFast EvaGreen
Supermix
Forward qPCR Primer (10 µM)
3.4
10
Reverse qPCR Primer (10 µM)
Diluted cDNA product
0.8
5
Total Volume
20
0.8
Comment
Primer concentrations may
have to be titered for optimal
performance according to the
qPCR reagents used
Table 4. qPCR Thermal Cycling Parameters
Description
Temperature (oC)
Time (s)
Cycles
Enzyme activation /
Initial denaturation
Denaturation
Annealing/Extension
95
30
1
95
60
5
30
50
Table 5. Troubleshooting Table
Problem
Large variation in Ct
observed between
biological replicates
Possible Reason
Inconsistent cell seeding
Incomplete cell
lysis/recovery
Cells in some wells may
have been washed away
qPCR “no template
control” shows signal
Contamination of reagents
or cross-contamination
during plate loading
Primer-dimer amplification
Possible Solution
Ensure equal numbers of cells are
seeded in each well by careful counting
and dispensing from a well-mixed stock of
cells in suspension.
Triturate cells through micropipette tip
more forcefully to ensure dissociation of
cell clumps.
Ensure complete recovery by scraping the
well with the micropipette tip, paying
particular attention to the cells on the
edges of the wells.
Wash cells gently with PBS, ensure cells
are not washed away by confirming
adhesion with a microscope.
Use dedicated micropipettes and filtered
tips to minimize contamination. If a cell
dilution is being assayed, ensure aliquots
are always done from lower to higher cell
concentrations.
Check and minimize primer-primer
hybridization using primer design software
Ensure hotstart DNA polymerase is used
for qPCR to reduce primer-dimer
formation.
No amplification
detected during
qPCR
RT reaction failed or
inhibited
RNase contamination of
labware
miRNA of interest could be
of low expression or is not
expressed in sample
Ensure that the RT is functional under the
lysis conditions. If lysis buffer is different
from what is suggested above, ensure RT
is able to proceed in the presence of
detergents used.
Lyse a serial dilution of cells and detect a
known, abundant small RNA or miRNA
(e.g. U6 snoRNA or miR-16) to validate
that RT is efficient.
Use dedicated micropipettes and filtered
tips for work with RNA. Do NOT use
micropipettes used for RNase work for
this application.
Use a higher (but not over-confluent) cell
density.
Validate assay with RT-qPCR on a serial
dilution of synthetic miRNA to determine
assay sensitivity.