Yeah, it would be crazy to play hide-and-go-seek with a GPS, night-vision goggles, and a heat-sensing camera. But that’s kind of similar to the pricey, sophisticated laboratory gadgets that researchers currently have to use to spot 2’-O-methylation hiding on RNAs. Now, scientists in China have come up with a much simpler, less expensive way (called RTL-P) to find these mods. The method doesn’t use radioactive labels and works well on rare or small RNAs. And it’s almost 1000 times more sensitive than other reverse transcriptase (RT)-based approaches.
“Reverse transcription at low deoxyribonucleoside triphosphate concentrations followed by polymerase chain reaction” (or RTL-P, for short) takes advantage of the fact that RT pauses at 2’-O-methylation. If dNTP concentrations are low, the enzyme usually stops cDNA synthesis at that spot. To figure out if a 2’-O-methylation is around, the team does the RT reactions at both low and high concentrations, amplifies the cDNAs with PCR, then compares the band intensities.
The primer for the RT reaction is downstream of the mod, so in low dNTP conditions, the researchers get mostly short cDNAs. In high dNTP conditions, RT doesn’t pause and stop, so they get long cDNAs. In the PCR step, one set of PCR primers amplifies the short cDNAs, and another set of primers amplifies the long ones. If there’s a mod around, then the band of short cDNAs is much more intense than the long-cDNA band on a gel.
The researchers also adapted the method to determine the specific location of a 2’-O-methylated site and to detect the mod at the 3’ end of small RNAs.
Get the long and the short of it by reading the details at Nucleic Acids Research, July 2012.