How many copies of the target sequence are in your 26ng DNA?

10000 copies should ideally give you a ct-value of around 23 to 24.

If you have only, say, 10 copies, you ideally won't get a ct lower than about 34.

Have you determined the amplification efficiency?

it should be close to 2. Lower efficiencies will not only shift ct to higher values but also make the entire PCR unreliable. If your problem are low efficiencies then there are two possible reasons:

- "bad samples" containing PCR inhibitors -> better/other purification protocol!

- "bad primers" -> design new primers

If you use a commercial PCR master mix, it is unlikely that the rest of the PCR mix is responsible. Some mixtures have low Mg++, and there you might add some additional Mg++ (to 4mM or even up to 6mM) to improve the reaction (balance with the amplification of unspecific products and primer-dimers).

How many copies of the target sequence are in your 26ng DNA?

10000 copies should ideally give you a ct-value of around 23 to 24.

If you have only, say, 10 copies, you ideally won't get a ct lower than about 34.

Have you determined the amplification efficiency?

it should be close to 2. Lower efficiencies will not only shift ct to higher values but also make the entire PCR unreliable. If your problem are low efficiencies then there are two possible reasons:

- "bad samples" containing PCR inhibitors -> better/other purification protocol!

- "bad primers" -> design new primers

If you use a commercial PCR master mix, it is unlikely that the rest of the PCR mix is responsible. Some mixtures have low Mg++, and there you might add some additional Mg++ (to 4mM or even up to 6mM) to improve the reaction (balance with the amplification of unspecific products and primer-dimers).

Hi Sunil,

I had the same issue with a low expression gene and I was able to circumvect it by using a TaqMan high capacity cDNA kit, starting with 2 ug of total RNA and using random primers. I did the reactions in 20 ul of final volume and used 2 ul of undiluted cDNA prep as template for the TaqMan Gene Expression Assay (this is the max amount of undiluted template that we can add according to the manufacturer's). In these conditions, I was able to pull a Ct of 23.

If your assay is reading the 5' of your mRNA, let's say at more than 3 kb from the polyA tail, I would suggest that you do the cDNA using a reverse primer specific for you gene, placed at about 500 bp downstream of your gene assay. That also solved my issues of low Ct in some of my other assays.

Finally, if you find out that your gene of interest has a GC rich region, you might need to use a thermostable RT (like Thermoscript RT-PCR system) that allows you to do the cDNA synthesis at 50-60 C and has a polymerase able to deal with some extent of GC-rich sequences. 

Hope this helps, wishing you successful Taqmans!

Hi Sunil.  Yes it completely sounds like your amplification efficiency is very poor.  In this situation, time and time again I see it is caused by poor template quality and/or bad primers.  I suggest you start by ensuring your DNA is good quality, using gel electrophoresis and nanodrop. Optimally use column based kits for extraction nucleic acids.  Next if still have poor amplification then check by in silico PCR that your primer/ probes really should work, redesign primers if appropriate, or get your regular primers remade if they definitely should be working. If still not working, then consider such "extreme" measures as different Taq, changing magnesium concentration, and annealing temperature.  One last thing, ensure you thermocycling profile is correct for your Taq mix - it's surprisingly common for people to make this mistake.

Thank you to all for very helpfull suggestion, Jochen Wilhelm sir, yes I am using commercial PCR master mix (  A Biosystem), I will try to optimize Mg++ concentration

Optimization makes little sense if you don't know (at least  by an order of magnitude) how many target copies you have in the PCR. If your sample material is cDNA, the expression of the gene might be very low. It might them be better to generate a reference sample (from purified conventional PCR) so that you can determine (and adjust) the copy number in the samples you use for optimization. If you do this, then take care of contaminations (tip: prepare the standard in a different lab and take only the used dilution of the purified and quantified PCR product back to your qPCR lab).

If the qPCR is them optimized and working well and robustly with the standard, *then* you can test using spike-ins of the standard if "real" biological sample material compromises the amplification efficiency etc.

Thank you Jochen Wilhelm sir