Biology Reference
In-Depth Information
Fig. 2
Schematic description of polyT adaptor-based miRNA qRT-PCR. Step 1:
Addition of a polyA-tail to any RNA molecule including miRNAs. Step 2: Reverse
transcription with an adaptor primer using an Oligo-dT with unique sequence at
the 5
end for universal reverse primer binding and a NV anchor (N: any base; V:
not T) at 3
′
end. Step 3: cDNA is used for qRT-PCR quantifi cation of mature
miRNA using a specifi c forward primer and a universal reverse primer (A) and for
precursor miRNA using specifi c forward and reverse primers (B)
′
2
Materials
Working with RNA is very demanding because of the chemical
instability of the RNA and the ubiquitous presence of ribonucle-
ases (RNases). These enzymes are diffi cult to inactivate, even after
prolonged boiling or autoclaving, they can maintain activity.
Therefore special precautions should be taken when working
with RNA:
Designate a special area for RNA work only.
●
Wear gloves at all times. After putting on gloves, don't touch
contaminated surfaces and equipment.
●
