Northern alternative

User: Wei Yan, University of Nevada, Reno Project: Probing temporospatial expression of small RNAs in testis. Problem: Northern blots are cumbersome and lack sensitivity, and RT-PCR isn't geared toward amplifying short nucleotide sequences. Related Articles Amplifying Trouble Tips for tricky PCR Staying clean Dilution Defixation Single cells

Josh P. Roberts
Apr 1, 2008

User:
Wei Yan, University of Nevada, Reno

Project:
Probing temporospatial expression of small RNAs in testis.

Problem:
Northern blots are cumbersome and lack sensitivity, and RT-PCR isn't geared toward amplifying short nucleotide sequences.

Solution:
The small RNAs in question — micro RNA (miRNA), small nucleolar RNA (snoRNA), and piwi-interacting RNA (piRNA) — can be as short as 20 nucleotides and lack poly-A tails typically associated with mRNA. Yan isolates the small RNA using Ambion's mirVana™ miRNA isolation kit. Then he polyadenylates the small RNA fraction, and reverse—transcribes the neo-poly-A RNA using a primer consisting of oligo-T and an adapter sequence.

The resulting cDNA is generally about 120 nucleotides long, Yan says, "a size we can use for regular PCR and for qPCR." The adapter sequence serves as a universal downstream PCR primer, while the small RNA...