Biology Reference
In-Depth Information
7. Due to the low RNA content in some manually microdissected
samples and the resulting low amount of isolated total RNA, it
is possible that the RNA needs to be concentrated (i.e., via
speed vac).
8. Amount of input RNA can be reduced to 1 ng; however, this
may reduce the sensitivity of your assay for low abundant
miRNA molecules.
9. Correct for losses during pipetting by increasing volumes in
your master mix by 10-20 %.
10. RT is possible in multiplex using more than one miRNA-
specifi c stem-loop primer in one RT reaction. We highly rec-
ommend controlling the results obtained from multiplex
reactions in a representative experiment using singleplex
qRT-PCR for each miRNA. Also include an appropriate
number of ncRNA molecules for normalization (
see
also
Note 12
).
11. If qRT-PCR may involve quantifi cation of mature and precur-
sor miRNAs in parallel, use the higher amount of RNA input
for cDNA synthesis for detection of mature miRNA as well.
12. Normalization is still an unresolved issue in qRT-PCR due to
the lack of endogenous molecules which are expressed at a
stable level in any cell/tissue type under all possible biological
conditions. Therefore, it is diffi cult to give a general recom-
mendation for a specifi c probe for normalization in miRNA
qRT-PCR assays. However, we recommend testing at least two
to three different ncRNA molecules (miRNAs or snoRNAs) in
your sample set in order to choose those molecules being most
stable under your experimental conditions.
13. We strongly recommend the use of NTC reactions to evaluate
background signal.
14. Keep all TaqMan
®
MicroRNA Assays protected from light,
excessive exposure to light may affect the fl uorescent probes.
15. Due to the initial incubation step at 95 °C during the PCR
reaction, it is not necessary to keep samples on ice during reac-
tion setup.
16. In addition, we recommend checking the specifi city of the
PCR product using standard gel electrophoresis.
17. The miScript Precursor Assay contains both a forward and a
reverse primer. Do not use the miScript Universal Primer.
18. In addition, we recommend checking the specifi city of the
PCR product using an electrophoretic gel.
