With Halloween coming soon, we usually expect some visits from goblins, ghosts, and other creepy creatures who aren’t really what they seem. Now, researchers warn that using commercial reverse transcriptase (RT) might give you another sort of ghastly scare—you might think you’re detecting antisense non-coding RNA (ncRNA) when it isn’t even there.
With so much recent interest in ncRNAs, more and more labs are looking for easy ways to detect them. One easy way is by performing RT-PCR, which is sensitive and doesn’t require fancy equipment or expertise. But when the researchers in France tried to use RT-PCR to find antisense ncRNA for RARβ2, they realized that they really got the sense strand, or mRNA, instead. They even tried it with different cells and different primers, but still got the same result. Doing the reaction without RNA or without RT gave no signal, as expected, but when they ran a no-primer control they were surprised to see a signal. After seeing the same nonspecific signal for several RNAs, the scientists think that it could be a global phenomenon.
So, what’s going on? The researchers did a little investigating and found evidence of short contaminating RNAs that are acting as primers. These contaminants stick tightly to the RT enzyme. Five commercially available RTs that were tested appear to have different levels of this contamination. The researchers say that, for most applications, this nonspecific background signal won’t matter. However, antisense ncRNAs are at such low levels that this background will likely be problematic in these types of experiments.
See all the frightening details at Biochimie, October 2011.