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lengthening the molecule by incorporating a poly(A) tail or stem-
loop structure [ 31 ]. Consequently, cDNA can be used in qPCR
using a miRNA specific set and one of the two major fluorescent
systems used for monitoring qPCR of miRNAs SYBR Green and
TaqMan probes. The main limitations of RT-qPCR include being
heavily time-consuming, especially in larger studies and being
prone to loss of specificity due to flanking primer overlap.
Before starting the experiment, RNA needs to be isolated from
the samples using a method that preserves small RNA species.
Subsequently, RT-qPCR is performed in two steps including (1)
conversion of RNA to cDNA by reverse transcription reaction and
(2) measuring of miRNAs using RT-qPCR.- Thaw the template RNA on ice.
- Prepare and label your tubes or 96-well plate ideally allowing
for comparison with a reference miRNA, controls and repli-
cates (see Note 11). - Thaw the required components on ice (see Note 12).
- Prepare the reverse transcription master mix by mixing
0.075 μL 100 mM dNTPs, 0.5 μL reverse transcriptase,
0.75 μL 10× Buffer, 0.095 μL RNase Inhibitor, 2.08 μL water,
and 1.5 μL of miRNA reverse transcription primer for both
miRNAs in polypropylene tubes (see Note 13). - Prepare four master mixes with a negative and a positive reverse
transcriptase batch for each miRNA (multiply the volume by
the number of wells for each corresponding well). - Mix and centrifuge briefly (see Note 14).
- Dispense 5.0 μL of reaction mix into each corresponding well.
- Dispense 2.5 μL of either experimental or reference miRNAs
into each 96-well reverse transcription reaction plate. - Seal the plate, mix and centrifuge briefly.
- Leave the plate on ice for 5 min.
- Keep the plate on ice until you are ready to perform the reverse
transcription reaction. - When ready, run reverse transcription plate on a 96-well PCR
platform, with sequential incubations at 16 °C for 30 min,
42 °C for 30 min, and 85 °C for 5 min. - Allow the plate to cool down and centrifuge for a brief time.
- Prepare and label your PCR-strips or tubes 96-well plate for
polymerase chain reaction. - Add 10.00 μL 2× Universal PCR Master Mix to 7.67 μL
Nuclease-free water for a total of 17.67 μL on ice into a poly-
propylene tube labeled PCR reaction (see Note 15).
3.3.1 Performing
the Reverse Transcription
Reaction
3.3.2 Performing
the Polymerase Chain
Reaction (PCR)
Moein Amin et al.