Authors
Rui Shi, Chenmin Yang, Ronald Sederoff, and Vincent Chiang
Summary
Here we describe a protocol for validating expression of artificial microRNAs (amiRNAs) by poly(A) tailing-based RT-PCR. Total RNAs, including amiRNA, are poly(A) tailed using E.coli. poly(A) polymerase. Poly(A) tailed amiRNA can be converted into cDNA along with mRNAs in a reverse transcription reaction primed by a standard poly(T) anchor adaptor. AmiRNA can then be amplified and quantitated by real-time PCR using an amiRNA specific forward primer and a universal adaptor primer. Because the amiRNA forward primer is designed without annealing to the 3’ end of the amiRNA, the precise 3’ end sequence of the amiRNA can be obtained by sequencing the PCR product and used as verification of the accuracy of expressed amiRNA. Poly(A) tailing-based RT-PCR also permits profiling of expression of amiRNA targets and other related genes with efficiency similar to that of standard RT-PCR.Further details
The protocol was published on Protocol Exchange on 3 February 2012. To see the entire protocol, click on the source link.Advertisement
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