Hello Kee

your basic description of things doesn't sound too bad. I concur with all of the above especially designing primers with programs like primer 3 plus and/or screening for things like hetero (primer) dimer with freeware like the IDT primer design tool or the Sigma equivalent

I will add a few more  things:

1. Verify your primer Tm using (for example) the IDT algorithm but remember to declare factors like primer conc dNTP conc and Mg concentration|: Duplex melting is a function in part of the local environment as well as intrinsic primer sequence and so by stating these parameters you will obtain a more accurate pertinent Tm

2. Screen primers initially by agarose gel electrophoresis: Try an annealing gradient from Tm-5C to TM +1 C and utilise the Tm yielding the most specific and efficient amplification in actual qPCR. This tends to be about Tm-2C so failing a gradient screen start by using this temp

3. For maximum primer efficiency in qPCR  and to hit that all important >/=85% make sure your amplicon is 100-200bp; no more than 300bp and preferably 100-200bp

4. With regard to primer situation bias your primer towards the 3' end of your cDNA and if priming with OligoDT alone keep your primer preferably

Hello Kee,

Are you using a good reverse transcriptase?  Hopefully not MMLV since that RT enzyme gives notoriously poor and differential yields of cDNA and, even denatured, can bind cDNA during the qPCR phase causing sporadic results.  Also, how are you isolating your RNA (by TRIzol or some other method)?  Your parameters sound good, but you should indeed do a pre-study of a dilution series of a mixture of your typical cDNAs from full-strength (1:10 diluted cDNA, 4 uL/20 uL qPCR; e.g. 1:50) out to 1:204,800 dilution of your cDNA.  Such a 13-pt serial 1:2 dilution study of your typical 1:10 prediluted cDNA mixture will tell you if you are diluting your cDNA out far enough for qPCR analysis. As is, the 1:50 dilution that you are now testing samples at (1:10 cDNA pre-dilution, and another 1:5 dilution incurred by putting sample in the qPCR), may not yet be enough dilution to avoid sample-introduced qPCReaction inhibition.  See the amplificatory behavior over a wide dilution series first, and then choose the dilution range window that gives you the best standard curve, and work within that range.  A singular point within the early part of your empirically-chosen standard curve range can then be used to examine all samples.  At first this all may seem counter-intuitive, but once you view the dilution profile, obvious things become more obvious.  The other possibility here (since you suspect primer dimer and nothing else at this point) is that you may need to re-check the sequences of your primers to be absolutely certain they have 100% fidelity for your target of interest; do not rely on previously published sequences to be accurate, ever. Search the data bases first, see with your own eyes where your primers are binding, and be sure the distance between the primers is suitable for the qPCR application, check the Tm values by at least 3 different algorithms/programs, and then decide which course of action to take.  Different sample isolates can inhibit the qPCR to different extents, but, once you've identified the dilution threshold after which this behavior is absent, you are in legitimate quantitative territory for all similarly-isolated samples. 

Thank you so much for your input! Can you recommend any programs that I can install to check the Tm and to evaluate the suitability of my primers for PCR? Thanks. 

I've heard that Primer 3 is OK.  And, also, if you go to the IDT web site (Integrated DNA Technologies) you can check Tm values there too.  I use primer Express version 2.0 or 3.0  (from Applied Biosystems - but, you have to buy one of their machines to get it for free - other than that it is $3000-$6000 unfortunately.  Bear in mind, all Tm calculating algorithms will not agree.  So, whichever one you choose, be sure to realize that.  Your goal is to generally achieve Tm = 60C, so, if the program says 58C or 62C you should still be OK -- e.g., close enough.

If you're worried: optimise your primers using normal PCR, followed by gel electrophoresis. It's much easier to distinguish primer dimers from a specific product via gel (ca. 20-25bp fuzzy blob vs 100-200bp neat band) than it is by guessing from melt curves.

I'd second using primer3, though: I have had good results using that.

http://primer3.ut.ee/

I typically design 2 pairs per gene (or more if necessary) and then test them first via conventional PCR against cDNA from a sample that SHOULD have the transcript and cDNA that absolutely shouldn't (and against water, too, just in case). A bonus is that you then get a PCR product you can purify and use in standard curves if needed. :-)

Hello Kee

your basic description of things doesn't sound too bad. I concur with all of the above especially designing primers with programs like primer 3 plus and/or screening for things like hetero (primer) dimer with freeware like the IDT primer design tool or the Sigma equivalent

I will add a few more  things:

1. Verify your primer Tm using (for example) the IDT algorithm but remember to declare factors like primer conc dNTP conc and Mg concentration|: Duplex melting is a function in part of the local environment as well as intrinsic primer sequence and so by stating these parameters you will obtain a more accurate pertinent Tm

2. Screen primers initially by agarose gel electrophoresis: Try an annealing gradient from Tm-5C to TM +1 C and utilise the Tm yielding the most specific and efficient amplification in actual qPCR. This tends to be about Tm-2C so failing a gradient screen start by using this temp

3. For maximum primer efficiency in qPCR  and to hit that all important >/=85% make sure your amplicon is 100-200bp; no more than 300bp and preferably 100-200bp

4. With regard to primer situation bias your primer towards the 3' end of your cDNA and if priming with OligoDT alone keep your primer preferably

All good advice above, I would only add that although it may seem time-consuming, it is reasonable to also test a range of input amount of RNA for cDNA synthesis and use the quantity that falls within the linear range (see the document attached, p.6) instead of the one that is recommended by the manufacturer. This ensures that all of your transcripts of interest are reverse-transcribed with similar efficiency and hence are well represented. Moreover, I would also recommend (unless it's a Taqman assay with a specific probe) to (clone and) sequence the PCR product for any SYBR green qPCR assay that has not yet been validated.

Good add on to my answer Olga !!