If I do a melting curve analysis for the PCR amplicons, for instance after the SybrGreen real-time PCR, I also see some differences in Tm between replicates. I checked if I can find a 0.8 degrees of centigrate difference in my old experiments and found one very quickly. The real-time PCR instruments are not so precise. For exact measurement of PCR products, people use High resolution melting (HRM) instruments. You do a little diffrent thing than me, because you measure primer melting, not amplicon melting, but I believe, the problem is similar.

Thank you for the comment on the issue.

Is it possible you have evaporation of some samples during the run? A change in salt concentration after evaporation will change the melting temperature. It has happened to me when I was using a plate with a film seal that was melting during the run.

Thank you. I was just wondering what could be the reason? I could further continue my work considering your reasons. could u suggest some tips to overcome this?

Ganga, I could see that the film was peeling off the plate in some areas and after spinning the plate down it was obvious that the volumes were different, so we switched to using lids instead. It depends on your system. If you see the volume is changing after the run check that you are securing the lids or film (or whatever you are using) as well as you can, then finally consider changing suppliers if problems continue. It could also be a problem with pipetting so I would suggest also checking by eye that there are no obvious differences between samples before the run.

Thank you. It would be of great help in minimizing errors while using RT.

i am again facing problems in standard curve for checking primer efficiency of target genes. I am facing high standard deviation error and getting multiple peaks at lower concentration, for example 0.2 or 0.02 ng/ul. Could the same dilution concentration is advisable for all target genes or i could change depend upon the target for calculating primer efficiency. Suggest some tips to avoid pipetting errors.

Pipetting errors mainly take time and practice to reduce. Changing tips each time and avoiding small volumes by making up master mix helps. Do serial dilutions in big volume.

When you say multiple peaks, do you mean the melt curve is showing more than one pcr product? This could be primer dimer, where if DNA template of your target is too low in concentration, the primers will start to amplify each other. You have to discard those points from your standard curve as the SYBR green will measure those too.

Yes, each target will be expressed to different levels in the cell so will not be accurately quantified when the levels reach a certain threshold. I run a standard curve with several dilutions and disregard anything at the top or bottom of the range that lowers the efficiency measurement of the standard curve. Then make sure your samples to test lie within the selected range.

thank you for the suggestions. The standard curve points could have duplicates or more than three replicate points for each dilutions. Then how many points is mandatory ? Right now i am using 5 points, among them only two points is efficiently touching the slope.

I would say aim for 3 points minimum to create your standard curve and do each point in triplicate. When you remove the bottom point, does that not change your slope to touch more of your points. The slope is calculated taking into account all of your standards, but if you know that the low concentration ones are wrong you should be able to remove these and the slope will be recalculated ignoring these points. It depends on what software you are using whether this works by just deselecting the points or if you have to tell the program that these samples are no longer standards. Your problem could well be pipetting error if you are not experienced though. Its not really normal to have such a low range of detection that only allows you to have two points. After removing the low points contaminated by primer dimer you should be able to get 4-5 orders of magnitude in your standard curve until your concentrations are so high they begin to inhibit your PCR reaction.

I could get four points if i would exclude replicate points. Since i am getting high standard deviation of 0.8 to 1 in some points, i excluded that points. I would follow up your suggestions and minimize my pipetting errors.

 Hi Ganga,

How did you cope with this problem, could you tell please? (regarding as a melting curve analysis for the primers, geting a different Tm for the same sample in the replicates)

The equal distribution of the samples in each replicates plays a major role in minimizing the difference Tm for same sample. You could give a quick spin of  sample and avoid pipetting errors. Hope this may helpful. 

Dear  Ganga Viswanath 

Could you please put a picture of the melt curve here, then I can help better.

Thanks

Dear Ganga,

There are a number of possible sources of variation in observed Tm, even when a single, clonal, amplicon is produced in a PCR.

A non-exhaustive list is as follows:

1) Thermal non-uniformity in the PCR machine block

2) Artefacts caused by the use of non-saturating fluorescent reporters e.g. SYBR Green

3) Variations in evaporative loss between tubes

4) Low optical sampling frequency when acquiring melt data

5) Low thermal resolution in the PCR machine

6) Variations in tube geometry and wall thickness

7) Noise in optical data

8) Noise in thermal data

9) Asynchronous acquisition of optical data

Best regards

Rod

All I can tell you is the gene expression patterns from tissue samples are different from.single cells isolated from the same tissue. one can detect different isoforms with different Tm curves during single cells, that is not detected or masked in total RNA isolated from tissue. This happens prrimarily because only few cells in a tissue make both the gene transcript and the isoform. one can use microfluidic rtpcr in order to see this phenomenon.