I have recently ordered the Taqman RNA-to-CT One-step kit in order to assess the expression regulation of 12 genes involved in the one-carbon metabolism and oxidative stress. Before starting with these RT-qPCR analyses, however, I decided to test the PCR efficiency at different concentrations of my RNA samples. I set up a serial dilution series (10ng/reaction – 160ng/reaction), picked one of the genes and fired away.
Yet none of my replicates corresponded with one another.
Immediately I questioned my pipetting skills. Therefore I asked two colleagues to perform the same PCR efficiency assay. But to no avail. None of the replicates aligned and the PCR efficiency ranged between 60% and 80%.
In the meantime, I have performed many RT-qPCR assays – with new and old reagents, with RNA from different tissues, different real-time PCR machines, with capped tubes and 96-well plates as well as purified and unpurified RNA. Still none of the replicates corresponded.
As the assays are far from repeatable, my work is quite at a dead end now.
Thus, I would like to know if someone has also encountered such a problem in the past? If so, what was the problem and how did you fix it?
Did you transcript your mRNA to cDNA ?
Have you checked your primers before you start?
What protocol you follow for running transcription of RNA to cDNA and RT-PCR?
Have you checked your mRNA sample purity?
I wish if get your answer.
All the best
Good evening Ali Saeed,
Thank you for your response.
The Taqman kit that I have used is designed to perform both the reverse transcription step and the amplification step in a single buffer system - in other words in the same tube. Therefore, the PCR cycle starts with a 15min step (at 48°C) where the RNA is supposed to be transcribed to cDNA. The amplification of the cDNA is then to follow directly after.
The primers that I have used are brand new and as they are from a household gene (ꞵ-2-microglobulin), our lab is familiar with its relative expression.
To this message I have attached the protocol of the kit that I've used.
Regarding the mRNA sample purity, I have recently tried out the MEGAclear Transcription Clean-Up Kit from Invitrogen in order to purify my RNA samples to a greater extent, but RT-qPCR results were comparable to the unpurified version of the samples.
Kind regards,
Liesel
Due to "One-Step" kit, you can not distinguish RT step with qPCR replicates. In a two-steps (RT and qPCR separated), technical replicates of qPCR is often very good because they depend only of your capacity to perform a good pipetting. Often, no replicates of RT is performed, we prefer doing biological replicates using the next cell passage or another individual.
When you do your one-step, you do RT replicates and not only qPCR replicates. Efficiency depends too of your RT efficiency and might be lower than expected.
In a two step protocol, I use 10 to 20ng/well of RNA but no more.
Does your CT is good and not too much high ?
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