Hi Tran,

efficiency depends also on the primer concentration, you should run a new calibration. It is possible that you are in saturating conditions and nothing will change, but you should make sure.

Why do you want to reduce the primer concentration? If you were always planning to lower it, why did you use a higher concentration initially?

Primers are cheap, so it can't be a money saving approach. Efficiency is an important factor in qPCR, so sticking with what works seems prudent.

Thank you very much Pietro Pilo Boyl

John Hildyard Thank for your recommend. The reason is that in the range of cDNA template 10 - 1000 ng, the Ct values I got were below 13 for the gene 16S (I'm studying on bacteria). However, for other genes, the Ct is often over 30 (even N/A) if I use

Huh. Ok, that is not how I would do it. At all.

You're suggesting that you make your PCR less efficient so you can get more reasonable Cq values? That won't actually be the outcome, what you'll effectively be doing is turning useful information into untrustworthy information.

Why not simply use less cDNA for the highly expressed genes?

I.e. prepare cDNA from all your samples, using the highest amount of RNA you trust your cDNA synthesis reaction to handle linearly. If a kit claims 10-1000ng, I would aim for say...800ng.

Dilute it as normal (I typically dilute all my cDNA 1/20 from the outset, simply because cDNA synthesis buffer is bad for PCR, so it helps to dilute it out, and even my lowest expression genes are still robustly detected at 1/20 dilution).

Then from your diluted cDNA, prepare a further set of tubes diluted yet again, perhaps another 10 or 20-fold (a 10-fold dilution will increase Cq values by ~3, and 20-fold by ~4). Use these for your ribosomal RNA qPCR, use your more concentrated cDNA stocks for your GOIs.

As long as you don't pipette terribly, these additionally-diluted stocks should still serve as entirely suitable reference sources.

John Hildyard Fortunately, my R square is always about 0,99 so I can skip the pipetting problem.

Thank you for your guidance, I will try some difference diluted cDNA.

However, is it ok if I use a smaller amount of template for gene 16S while keeping the volume of the others? e.g. 1 ng cDNA for 16S (reference gene) and 50 ng cDNA for the other genes? Could the result be used for calculation the fold?

Your cDNA quantity will always be an estimate (cDNA synthesis may or may not be 1:1 conversion), so saying "1ng cDNA" isn't strictly accurate.

Other than that, what you are suggesting is EXACTLY what I am saying: make your cDNA, then dilute accordingly. E.g. make a cDNA prep and dilute 1/20.

Use 2ul of that for your GOIs.

Take 5ul of that prep and add to 495ul H2O (so now 1/1000)

Use 2ul of that for 16S.

The important thing is to use the same stock cDNA prep for both (so don't make two separate cDNA preps with different amounts of RNA).

John Hildyard I understood. Thank you very much for your great help.

Hi again Tran,

it might be convenient to choose a different RNA for calibration... of course rRNA is among the most abundant species, in bacteria probably the most. But maybe another mRNA for some metabolic enzyme might allow you to increase the template cDNA, obtain Cts for target genes below 30 and still be around 20 with your reference... Besides, mRNA in bacteria is very short-lived, while rRNA more stable, this might become source of variability... keep an eye on it.

Pietro Pilo Boyl Thank you for your advice. I will search for some other reference genes.