"increasing cDNA from 5 to 50ng the cts "

Do you mean the RNA input because cDNA cannot be quantitated this way and i noticed this is becoming a trend. I worked with cardiac tissue and i wouldn't go below 500 ng input RNA.

I wouldn't design primers with more than 5*C difference.

I used qPCR cycling conditions of an initial denaturation at 95°C for 3 minutes followed by 40 cycles of 95°C for 15 seconds and 61°C for 60 seconds. After the final PCR cycle, reactions underwent melt curve analysis to detect non-specific amplicons.

Also what was you cDNA dilution?

"increasing cDNA from 5 to 50ng the cts "

Yes the rna input, ct 27 and 24, respectively. This is for housekeeping for other genes ct is coming up in 30s. At 5 and 50ng.

My sybr kit says no more than 100ng of template. And given that I need to quantity 14genes, duplicate reactions that would be too many cDNA synthesis reactions (Economically, speaking).

Cdna 1is to 10 dilution.

The problem is I have diluted my cDNA and set nicely in alliquotes for use. My each ul has 5ng. I guess I can increase to 20ng max by picking more volume.

Dear Sulail

• Initially extract my cardiac RNA with Trizol; Chloroform and further extract the aqueous layer using a RNA purification column
• In my experience this culminates in the best quality total RNA eith the highest 260/280 ratio ( > 1.8) and the highest 260/230 ratio (>> 1.0; > 1.5)
• Having extracted your RNA as above I would use 200ng in the RT reaction and I would reverse transcribe for 1-2 hours (not 15 min as some kits recommend)
• In terms of qPCR conditions I would use conditions where the annealing temp is Tm-2C t0 Tm-5C: I would actually do that by initially gel based RT PCR, using an annealing gradient from Tm-5C to Tm in 1C intervals, i.e. 5 x PCR reactions for each primer pair
• Pick the annealing temp that gives the strongest band on a gel without contaminating (non specific) peaks
• Then verify that if you carry out a qPCR reaction that you just end up with a single strong peak in a melt curve and not extra (non specific) peaks of any significant size (compared to your main peak)
• I would also carry out a preliminary melt curve screen as described using a small range of primer concentrations for this one annealing temp: That is 1ul of 2uM primers (=2 pmoles of each primer); 1ul of 5uM (=5 pmoles) and 1ul of 10uM ( = 10 p moles of primer)
• You can use either 2 step or 3 step cycling parameters in your sybr green qPCR but providing your primers have Tm of 60-65 C then a combined annealing extension of 60C tends to be good but verify that by a preliminary gel based screen and subsequent sybr green melt curve analysis: So by way of example, if your primers had Tms of 60C and your optimal gel based annealing temp was 55C, then you would carry out a 3 step sybr green qPCR reaction in which you annneal t 55C and extend at 60C (trying a combined annelaing and ext @ 55C would render extension too inefficient, generally speaking)
• Perform your initial PCR incidentally with 1ul of 10uM primers : Generally, because qPCR is ~ 10 x more sensitive you tend to use routinely ~ 2uM of primer stock (1ul/rxn) but titrating upwards can help with CT values of low copy number genes
• Also add 2% DMSO to both your RT and PCR/ sybr green qPCR reactions

Thank you Laurence for your comprehensive reply. What is your suggestion about increasing denaturation time and elongation time (2step pcr)?

I am using 30s initial denaturation followed by 5 sec denaturation in cycling stage for 40 - 45 cycles (which I shall trim down to 35). Furthermore, elongation time I am using is 30s. Can increasing initial denaturation time and cycling elongation type help?

what is your experience with MgCl2?

Dear Sulail

• For short qPCR products increasing denaturation and elongation time in both the context of 2 step and 3 step qPCR cycles is unlikely to help
• However adding 5% DMSO to your reaction in conjunction with standard annealing and extension times can help
• Regarding the single initial hot start/denaturation cycle before commencing your 40 cycles of 2 step or 3 step PCR, take a look at the base composition of your intended target. Is it > 70% GC rich and/or contain runs of 4 or more G/C residues in places ? If so denaturing at 96-98C and doubling the time of the initial hot start cycle can sometimes help (in conjunction with 5% DMSO)
• Regarding MgCl(2) yes that boost the activity of your AmpliTaq/Enzyme formulation by increasing but can also lead to non specific amplification:
• Thus, if you find your Ct value is > 30 cycles, add an extra 0.5mM MgCl(2) to your sybr green qPCR reaction. This will potentially reduce your Ct value but runs the risk of non specific amplification, i.e. an extra peak in the melt curve
• Finally, Make sure your target region for your qPCR primers is within 1-2kb of the Poly A tail of your mRNA GOI and preferably at the last exon-exon junction or within the 3' UTR: That will result in more efficient RT and subsequent sybr green PCR

I did little playing with annealing temperatures & DSMO. The tm of my primer is 62.5 & 62.9, F& R, respectively.

1- With DSMO my Cts went higher from 33 to 35 tried with ta 60.

2- Without DMSO, when I checked at ta 59, my Cts were in the range of 25-30 (tm of product at 83). But this came at the cost of a positive signal in NTC at Ct 36 (tm of product at 76.6). Out of my 40 samples 7 samples had a small peak before my product peak.

3- Without DMSO, at ta 60.5, I had no positive signal in NTC but all CTs were between 30-36.

So, in your eyes. Is it good to have low CT at the cost of some NTC signal or relatively higher CT with clean NTC and melt curve.

Another thing, will it be acceptable if I run my target and housekeeping genes in separate runs. My housekeeping has tm for 64.5 and my 14 target genes have variable tm too. Optimizing gets messier and running housekeeping each time is not economical for me.

Thanks Laurence!

This is for one of my samples giving ct between 25 and 30.

p.s. Sorry for the poor quality capture.

You also have to keep in mind the PCR efficiency, which to some extent will be more important than the Cts.

Dear Kiessling, efficiencies were 94.5% for these runs. i am confused which is better high ct with nothing in ntc or lower ct at the cost of positive ntc signal at the last of my cycles. I ran 40 cycles. Do you think if I run 35 cycles my ntc will have no positive signal? all my samples showed ct less than 30.

Your melt curves aren't normal so unfortunately i wouldn't say that 94.5% is a reliable measurement. Can you show the NTC melt curve and amplification plot?

One of them is sample with two peaks one is ntc with one peak. My apologies the captures are bad and I m not in lab now.