Biology Reference
In-Depth Information
It is strongly recommended to perform a melting curve
(70-85 °C, read every 0.5 °C for 3 s) analysis at the end of the
cycling program (
see
Note 18
).
3.9 Relative
Quantifi cation
of miRNAs
Relative quantifi cation is the most commonly used analysis strategy
determining changes in steady-state gene expression levels in a
given sample relative to the expression of a reference gene. The
data output is expressed as a fold-change or a fold-difference of
expression levels. We recommend to calculate the relative expres-
sion ratio of a target gene using the method developed by M.W.
Pfaffl [
5
]. PCR effi ciency can be established either as described by
Pfaffl et al. or using algorithms such as implemented in the
LinRegPCR software [
6
] (for more detailed information refer to
the specifi c pages on
http://www.Gene-Quantifi cation.info
edited
by Michael W. Pfaffl ). A more simplistic approach is the so-called
“delta-delta Ct method” (Applied Biosystems) which is based on
the assumption that real-time amplifi cation effi ciencies of both tar-
get and reference gene are close to the ideal effi ciency of 2 (the
amount of PCR amplicon is doubled in each PCR cycle). The
known strong impact of differences in primer effi ciencies on the
calculated expression results should restrict the usage of this
method to quick estimations only.
4
Notes
1. For a more detailed protocol please refer to [
4
].
2. Manual microdissection is used because, in our hands, the
procedure is easier and faster than using laser capture microdis-
section, besides this technology (LCM) presents challenges,
particularly with regard to isolating intact RNA for downstream
applications.
3. If necessary, samples could be stored in lysis solution at −80 °C.
4. Make sure that the sample solution is clear otherwise disrupt
the sample using a micropestle. However, try to avoid this step
because this will reduce the yield of RNA quantity.
5. Although the RIN facilitates the assessment of the RNA qual-
ity, it should not be taken as an absolute tool for determining
the quality of RNA. As shown in Fig.
3
the RIN does not nec-
essarily refl ect the exact level of RNA degradation.
6. The amount of isolated RNA should be initially defi ned by
using a spectrophotometer in order to avoid the overloading
of the RNA LabChip
®
. In addition, the sample concentra-
tion read from the BioAnalyzer should be regarded as
semiquantitative.
