Hi Adela,

dilution of primer is not the best idea since your qPCR might run out off linearity. If you are not restricted to 18S, use other reference genes like actin or a ubiquitin. The second issue occurs if your RT-mix contain oligo-dT primers for reverse transcription the 18S is not efficienctly reverse transcribed.

If you are restricted to 18S you can use dilution series of cDNA to check for linearity of your qPCR signal and primer efficiency.

Best wishes

Lars

Adela, if you do use 18S as a ref gene for your qRT-PCR, prior to use, you will want to be sure to pre-dilute its samples at least 1:500 more than you dilute your samples for evaluating your other targets of interest.  If you do not, you will template-inhibit the RT and PCR phases of the qRT-PCR. 18S has a very wide dynamic dilution range since it is so abundant; in RT-qPCR, its log-linear amplification range commonly lies between 1:5000 to 1:5,000,000 dilution, or further. It can indeed be used (if found to be a stable ref gene up front), but samples need much higher pre-dilution before use.

In agreement with Lars (above), dilution of primers would not be the main issue for assessing 18S; it is the appropriate pre-dilution of the samples that will be key.

Thank you very much! After experiment with dilution series I found the best dilution of samples is 1:2500, thanks a lot again, Adela

Excellent - that sounds perfectly reasonable Adela~

Dear Jack,

Is there any published paper on this issue? We are working on viral RNA amplification from total RNA and we are also facing template inhibition, I think. will the same dilution of the total RNA enhance viral RNA detection?

SD

If isolating total RNA from lung tissue (infected with virus), a pre-dilution of at least 1:120 seems to bring the viral signal into its log-linear amplification range. This is for RSV anyway.

My above comment refers to TRIzol-isolated total RNA from 0.1 g of lung tissue in 1 mL of TRIzol.  After which the total RNA pellet is resolubilized in 170 uL of nuclease-free water.  Then 70 uL of that is TURBO DNAse-treated in a final volume of 110 uL. Then 80 uL of that supernatant is diluted 1:10 in 10 uL RNAseOUT and 710 uL nuclease-free water.  Then, that solution is spec'd against the appropriate blank, and then, that sample is diluted such that 6 uL of it in a 25 uL RT-qPCR reactions attains 0.78441 ng total RNA/uL in the RT-qPCReaction itself.  In the end, the effective dilution of the sample (including the dilution incurred by placing it in the RT-qPCReaction itself) is near or more than 1:500.  But, prior to RT-qPCR, at least a 1:120 dilution has occurred.

Another possibly helpful self-spun diatribe - attached.