Hi Sladjana

There are many reasons of high CP values. As you said, one could be that the amount of cDNA is very low (real low level of transcripts or low quality of cDNA). Another issue related to high CP values could be the presence of PCR inhibitors in the samples. In that case, to solve this problem you need to dilute more (e.g. 1:10) the cDNA.

Did you synthetize the cDNA by using oligo-dT? The oligo-dT initiate the reverse transcription at the 3´ end of the transcript. Secondary structure may lead to incomplete cDNA synthesis. In some cases, especially if the specific primers are designed near the 5' end, only few complete cDNA will be amplified. One possible solution could be to use random primers insted of oligo-dT.

The presence of multiple peaks could be due to the presence of aspecific amplicons, degraded cDNA, or presence of primer dimers. In RT-qPCR the concentration of primers should be lower than in PCR (3-5 pmol per single reaction). Higher amount of primers could lead to the formation of dimers (or even concatemers), that are visualized as multiple peaks in the melting curve (generally lower than the peak of the transcript).

How do you check if the genomic DNA has been removed from the cDNA samples? Contamination of gDNA in cDNA could lead to the formation of multiple amplicons which differ in length.

Last but not least, transcript isoforms could also lead to formation of multiple melting peaks. In that case, a possible solution may be to design a new set of primers.

Hope this helps!

Hi Sladjana

There are many reasons of high CP values. As you said, one could be that the amount of cDNA is very low (real low level of transcripts or low quality of cDNA). Another issue related to high CP values could be the presence of PCR inhibitors in the samples. In that case, to solve this problem you need to dilute more (e.g. 1:10) the cDNA.

Did you synthetize the cDNA by using oligo-dT? The oligo-dT initiate the reverse transcription at the 3´ end of the transcript. Secondary structure may lead to incomplete cDNA synthesis. In some cases, especially if the specific primers are designed near the 5' end, only few complete cDNA will be amplified. One possible solution could be to use random primers insted of oligo-dT.

The presence of multiple peaks could be due to the presence of aspecific amplicons, degraded cDNA, or presence of primer dimers. In RT-qPCR the concentration of primers should be lower than in PCR (3-5 pmol per single reaction). Higher amount of primers could lead to the formation of dimers (or even concatemers), that are visualized as multiple peaks in the melting curve (generally lower than the peak of the transcript).

How do you check if the genomic DNA has been removed from the cDNA samples? Contamination of gDNA in cDNA could lead to the formation of multiple amplicons which differ in length.

Last but not least, transcript isoforms could also lead to formation of multiple melting peaks. In that case, a possible solution may be to design a new set of primers.

Hope this helps!

• You can go as low as 1uM primer in qPCR; especially for high copy number genes
• This will reduce the likely hood of non specific peaks, especially primer dimer, but will also reduce your specific signal
• To elucidate non specific bands linked to primer dimer use a no template control (NTC) in your qPCR: This includes water in stead of cDNA in your sybr green qPCR reaction mix
• To address genomic contamination which could lead to specific peaks by qPCR but not necessarily on a gel if < 10ng ( Distinct bands are reliably detected on agarose gels to ~ 20ng whereas sybr green qPCR can detect bands to < 2ng; being ~ 10 x more sensitive), you need to have a no RT control (if at all possible, where you use water instead of cDNA during RT)
• To address specific but 'by'  products like pseudo genes and alternative transcripts BLAST your primers to verify specificity
• If you are not already doing so, make sure you design your primers across exon boundaries using ref Seq models in GenBank; Primer BLAST is ideal for that:
• https://www.ncbi.nlm.nih.gov/tools/primer-blast/
• I have attached a protocol detailing how to access Ref Seq transcripts in Gen Bank and then make exon (splice form) specific primers

Dear Sladjana, typically there is nothing much to say after the above opinions of dedicated experts, I would just like to contribute some facts by my personal experience in this iceberg, may be something clicks in your mind. This issue itself is so huge and has multiple dimensions to understand and to work out properly to solve the headache.

Usually for a Sybr Green assay 200 – 900 nM range of primers is very fine to work with, as this concentration avoids the formation of primer dimers and also inhibitory effect of SG dye on PCR is nil at this level, its ok theoretically, but I used to work even with 2500 nM (2.5 pmole / uL) primers with at least 25 ng cDNA in various studies without ever nailing down.

This actually all starts from RNA extraction step, if you got good RNA which I mean integrity wise not just the A260/280 based wonderful exciting readings of nanodrop, then  no doubt you have good cDNA in your hand as the latest cDNA (RT) kits have the mixtures of OligoDTs  along with random hexamers or decamers to sort the issues of cDNA synthesis  from the RNA type you have after extraction. So really here no need to worry much for this step. Main problem often comes from an issue, which many people don’t take with much seriousness and this is hidden in the long term storage of cDNA samples at -25 C and then multiple freeze thawing of such samples mainly the diluted ones so often same Ct readings won’t appear with the same sample and same primers in next time.

Primer degradation also increases the PD formation so you can make fresh dilutions from your main stock of GOI to avoid any possibility of this.

Primer designing is one of the main problems I believe in, as most of such strange issues of multiple peaks are coming by non-specific improperly designed primers only, so always design by at least targeting three different regions of GOI to get 3 primer sets of a same gene, and it really helps a lot.

Well known facts about the increased Ct variations (right shifts mainly) are low copy targets, the assay design, which does not detect all of the splice variants for that GOI, and increased primer dimer formation, which always delays the Ct, also the diluted cDNA and increase inhibitors in your sample are usual factors for a delayed Ct. Over a Melt Curve, gDNA peaks, PD peaks, possible spliced variant peaks and GOI product peak all have various behaviors to recognize that which situation is actually prevailing in your reaction. So in a melt curve, we always compare the amplicon peak Tm with the Tm of other peaks to reach some conclusion and that’s the beauty of melt curve analysis. The gDNA peaks usually comes after the GOI peak because of gDNA larger size, while this gDNA presence brings the Cts earlier as well than expected.

That is why some researchers suggest that after the elongation step of your qPCR cycle just add a temperature which is more than the primer dimer (which you suspect from the peak behavior), and less than the Tm of your product or GOI to get maximum signal solely from your target. But primer dimers after the Ct of GOI are not really significant.

Due to these multiple issues, we calculate and consider only the fold change (delts-delta Ct) rather than looking at raw Ct values and to get ourselves into worries.  As delta Ct values are independent of sample recovery and remains almost the same whether cDNA input varies (increases or decreases) among replicates to some extent so its the actual way in fact.

As per troubleshooting guidelines, if your PCR efficiency is less than 80 % so design proper primers and if the efficiency (E) is above 110 % so mainly by gDNA contamination, primer dimers and improper dilution workout.             Regards……

Dear Sladjana

i have decided to add to and qualify my first answer having reread your question and in light of other informative answers especially Jawads:

• Generally speaking lower Cp(t) values reflect lower gene copy number of your cDNA
• As mentioned by Jawad you can maximise your cDNA synthesis efficiency to offset this to some extent by using random hexamers during cDNA synthesis 
• I would add to this by saying that if at all possible reverse transcribe from 2ug total RNA (if copy number is known to be low); replicate your parent RNA for 1-2 hours (not 30 min as sometimes recommended) and try and use a high processive  RT enzyme which reverse transcribes at >50C like superscript IV
• What is more add either 5% DMSO to both your RT reaction and subsequent qPCR
• Alternatively and better still add 1-2 M betaine by diluting a 5M starting stock:
•  http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0011024
• http://www.sigmaaldrich.com/catalog/product/sigma/b0300?lang=en®ion=GB
• On reflection I would say that reducing your primer concentrations will not improve the situation with extra melt peaks but rather increase your Cp values still further. Thus the solution to these extra peaks is alternative specific primers designed according to the doc attached to my last answer 
• Irrespective, does it matter if my Ct values are low reflecting low copy number ? In short providing your Ct values are less than 35 and triplicates are +/- 0.5 cycles of one another the answer is no
• Futhermore, if you dilute your cDNA to generate standard curves and your R2 correlation coefficient is > 0.95 then you are within dynamic range and data is valid for expression analysis
• What is more standard curves will tell you if your primers have efficiencies > 80% and are thus applicable to expression analysis by the Livak method and also that inefficient amplification is not contributing to your low Ct values 

Did you check if the RNA used was ok? I mean, if is not degraded?