known inhibitors of reverse transcriptase in worms?

Hi All,

My lab has been experiencing problems with low efficiency in qRT-PCR reactions using cDNA prepared from total RNA isolated from synchronized populations
of worms. We follow the typical protocol of RNA isolation from worms (Trizol, chloroform extraction, isopropanol precipitation and ethanol wash) as
published in wormbook, etc. At this point we have tried independent cDNA preps and three different pairs of pubilshed primers, including primers for
tubulin and all have an efficiency of about 60%.

I’m wondering if there may be something in our RNA preps that is inhibiting the reverse transcription reaction. We get good A260/280 ratios from our
RNA preps, and rRNA bands are robust when we check the quality of RNA by agarose gel electrophoresis, so the integrity of the RNA itself seems OK.
Any ideas on if there’s something specific to worms to watch out for that could be resulting in low efficiency in our reactions? Many thanks!

I’m unaware of any worm-specific inhibitors of qPCR, but a number of non-specific factors have been reported. The Trizol interface contains contaminants that can inhibit qPCR, so it’s critical to avoid while pipetting the aqueous phase. Other inhibitors include glycogen (sometimes added as a carrier) and high rRNA/tRNA concentrations (from using excessive RNA template in the RT reaction).

The simplest solution is to make serial dilutions of your samples, perform qPCR, and identify the range of dilutions that yield linear results (i.e., dilute the inhibitor to the point where it no longer affects your assay).

Could also be your RT kit itself is off?