Hi Anna

Welcome back to the bench! With RT-PCR, the method we routinely use in the lab for quantification is the ddCt method. This choice of analysis needs to be fed in the RT-PCR machine (Viia7 Applied Biosystem is the one we have here).

We screen Melting curves to ensure that the reaction per well has a single product, symbolized with a single peak on the melting temp graph. We exclude the wells with multiple peaks and negatives which present weird peaks.

For example: If a well with NTC + cDNA gives multiple peaks, it cannot be used for analysis.

If for a primer set, the NTC+ cDNA control (cDNA reagents mix with no RNA) gives a single peak ,there is likely contamination with another DNA like product, hence the entire primer pair set requires repetition.

There may be differences in how different researchers prfer to exclude reads by melting curve observation.

Hope this helps

Best

Aparajita

Dear Anna!

To avoid the most common troubleshooting in the RT-PCR / RT-qPCR techniques, please visit the below link that would be helpful.

https://www.sigmaaldrich.com/technicaldocuments/articles/biology/troubleshooting.html

Hi Anna,

always record also the melting curve and add in each reaction a tube with all reagents + water instead of cDNA, which will give you a good indication in the melt curve. Also, consider that this is still a PCR, if you have doubts about the reaction, recover the plate after the PCR, add loading dye and run a gel, if you see a band at the expected size, it means it worked! :) Also, make sure that you designed primers to be on exon-exon boundaries, to exclude genomic contamination, and use some RT to test the efficiency of the primers with a titration curve. You might find this give very helpful https://www.thermofisher.com/content/dam/LifeTech/global/Forms/PDF/real-time-pcr-handbook.pdf

Ibrar Muhammad Khan, the link gives Error 404. Can you write the article title, please?

Dear Aparajita Lahree ! Thank you for your kind words))

Need to detalize: by NTC I mean water+ EvaGreen, primers mix (without cDNA). Apparently, you mean something else. Please, explain, what are:

1. NTC + cDNA?

2. NTC+ cDNA control?

You exclude: samples with multiple, weird peaks of melting curves, right?

Best,

Anna

Hi, Michele Gabriele !

I add NTC = water+ EvaGreen, primers mix (without cDNA) for each experimental/control group.

Is there any algorithm of designed primers to be on exon-exon boundaries?

Thanks for a useful link.

Anna

Hello Anna!

By NTC I mean SYBR master mix+ water+primer

cDNA refers to the cDNA reaction (this contains all the components of first strand synthesis)

cDNA control refers to the blank first strand synthesis master mix (no RNA added)

In the RT-PCR plate : we have a control with only the NTC mix and a control where there is NTC + cDNA control (to account for impurity coming from the first strand synthesis rxn)

of course for each primer pair the NTC changes. Both these are negative controls and should not show a single nest peak but rather a zig zag non specific pattern or nothing at best. The presence of any peaks in these controls means one needs to repeat that group.

Hope this helps

Best

Aparajita

Aparajita Lahree, thanks a lot! Yes, it helps)