Biology Reference
In-Depth Information
cDNA synthesis and qPCR. Both methods are detailed below. Reactions should be
done in duplicate or triplicate to reduce erroneous results due to pipetting error. A
nontemplate control reaction should also be included and a dissociation curve should
be calculated to determine if nonspecific primer interactions occur.
1. Primer Selection
Primary miRNAs can be amplified by selecting primers near to but not included in
the pre-miRNA hairpin, while precursor miRNAs can be amplified by selecting
primers within the hairpin. Since all pre-miRNAs are contained within pri-miRNAs,
the origin of amplification occurring from pre-miRNA hairpin primers cannot be
specifically assigned. However, comparison of signal from pre- and pri-miRNA
primer sets can determine the amount of pre-miRNA signal derived from pri- or
pre-miRNA (
Van Wynsberghe et al., 2011
). Primers should have a Tm between
58 and 60
C, a GC content between 30% and 80%, and the 5 nt at the 3
0
end of the
primer should have no more than two G or C nts. Amplicons should be less than
200 nt. Analysis of mature miRNAs by qPCR presents additional problems due to
their small size, but can be accomplished by adding linkers to the mature miRNA
(
Benes and Castoldi, 2010
). Below we detail methods for analyzing pri- and pre-
miRNA levels by 1 and 2 step qPCR.
2. DNase Treatment
RNA prepared as described above in Section I is generally devoid of DNA
contamination, but a DNase treatment of total RNA is recommended before RT-
PCR. This step can be performed between the first Trizol RNA extraction and the
second phenol extraction as described above in Section I. To the RNA resuspended in
200
m
L of DEPC water add 10
m
L of RQ1 DNase and 23
m
Lof10
RQ1 DNase
buffer (Promega). Incubate at 37
C for 1 h before adding 1
m
L of stop solution
(Promega). Proceed with second RNA phenol extraction as described above in
Section I.
3. One-Step qRT-PCR
The amount of total RNA and primers used in this reaction may need to be
optimized depending on the abundance of the particular transcript being analyzed.
In general 0.5
m
g of total RNA and 6.25 pmol of both forward and reverse primers are
added to a minimum 20
m
L reaction volume containing 1
RT Enzyme Mix, 1
RT-
PCR Mix, and the Taqman probe if performing a Taqman reaction (Applied
Biosystems). Increasing the reaction volume will decrease pipetting error but
increase the cost per reaction. Many companies sell kits or individual reagents for
qRT-PCR including Applied Biosystems and Bio-Rad. Reactions are run in a qPCR
thermocycler set to perform the following protocol: (1) 30 min at 48
C, (2) 10 min at
95
C, (3) 40 cycles of 15 s at 95
C and 1 min at 60
C, and (4) hold at 4
C.
Search WWH ::
Custom Search