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.

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 sequence itself gives specificity to the reaction as the upstream primer. Profiling hundreds of small RNAs by Northern blotting, even if it were sensitive enough, would "kill a student," he remarks.

When Yan started the project, there was only one method described to prepare small RNAs for PCR: a looped RT primer kit from ABI. Because the company didn't reveal the sequence of the looped primer, using the method required buying the kit, and "that really annoyed me," says Yan. Not long after Yan published his own method, though, several other companies introduced kits that do essentially the same thing, but still at a much higher price per reaction than his homebrewed version.

Cost:
Seungil Ro, codeveloper of Yan's homebrew method, estimates the cost at $8 per reaction, including $6 for the gene-specific primer. They get approximately 500 samples per reaction. Ambion's mirVana RNA isolation kit costs $220.