For qPCR is recommended to use 50 cycles, so you have a complete curve and get better info when using softwares that analyse raw data, however, any amplification or any Ct that appear after the cycle 37 it is very possible that will be a contamination. The most common contamination in the negative control is PCR product, and assuming that you are using filtered tips, good practices and all that, there is one factor that is there and we don't see, and it is the PCR product that is aerosolized in the air, typical appear in the new labs after 6 month of work. The source, is when you open a PCR tube in the lab to run a gel or to do a dilution curve etc... So one easy solution is never open a PCR tube in the lab (do it in the fume gas cabinet) and if you google you find webs sites with excellent recommendations about it (http://bitesizebio.com/19880/the-devil-is-in-the-details-how-to-setup-a-pcr-laboratory/  or   http://bitesizebio.com/20773/clean-up-your-act-how-to-clean-up-pcr-contamination/).

But please never run a qPCR with less cycles, this is a bad practice that make our work more difficult (I work in qPCR algorithms to analyse raw data) haha. 

Hi

I suppose you are using SybrGreen. Did you check the melting temperature of the PCR product?. It might be you are having unspecific amplification or primer dimer. Both gives you an amplification curve. but, checking the melting temperature, it may be completely different.

At the end of the PCR run, add a cicle of "Melting" and, as you do not know the melting temperature of your amplicon, use a wide range of temperatures. try to  use an interval from 50 to 95 ºC.

I hope it had help

Eugénia

Hi, Thanks for advise, YES I checked the melting temp and its same for both test and negative control. even the CT value is same in qPCR. 

I checked other possibility already by using genomic DNA in normal PCR, whereas there is size difference in pcr product. (negative control always gives amplification of cDNA pcr product size)

thanks

Check to see if your primers are specific for mRNA (e.g. via intron-spanning), and check to see whether they are specific to your gene of interest by in silico PCR (https://genome.ucsc.edu/cgi-bin/hgPcr). If the primers are not specific for RNA, you might be able to reduce genomic DNA amplification by DNAse'ing your RNA samples. Alternatively, you could re-design the primers.

Hello!

Try to decrease the PCR cycles

Hi Brian Bower,

thank for your advice, its also interesting to learn this, unfortunately this in silico PCR did not accomodate plants/Rice. and i am working on rice.

For qPCR is recommended to use 50 cycles, so you have a complete curve and get better info when using softwares that analyse raw data, however, any amplification or any Ct that appear after the cycle 37 it is very possible that will be a contamination. The most common contamination in the negative control is PCR product, and assuming that you are using filtered tips, good practices and all that, there is one factor that is there and we don't see, and it is the PCR product that is aerosolized in the air, typical appear in the new labs after 6 month of work. The source, is when you open a PCR tube in the lab to run a gel or to do a dilution curve etc... So one easy solution is never open a PCR tube in the lab (do it in the fume gas cabinet) and if you google you find webs sites with excellent recommendations about it (http://bitesizebio.com/19880/the-devil-is-in-the-details-how-to-setup-a-pcr-laboratory/  or   http://bitesizebio.com/20773/clean-up-your-act-how-to-clean-up-pcr-contamination/).

But please never run a qPCR with less cycles, this is a bad practice that make our work more difficult (I work in qPCR algorithms to analyse raw data) haha. 

It's a shame about the lack of an in silico PCR option for Rice. For what it's worth, the Roche UPL primer design tool can be used to generate intron-spanning primers for rice (https://lifescience.roche.com/webapp/wcs/stores/servlet/CategoryDisplay?tab=Assay+Design+Center&identifier=Universal+Probe+Library&langId=-1). These are intended for use with their Universal Probe Reagents, but the primers can also be used with SYBR green reagents. IDT's qPCR design tool may also work, but it doesn't have a way of explicity specifying organism at the start of the primer design process, and it doesn't seem to recognize some Rice Accession IDs (https://www.idtdna.com/scitools/Applications/RealTimePCR/).

Hi Juan Pablo Matte Risopatron, and Brian Bower

Thanks for the answer, its true that aerosols can make problems or even number of qPCR cycles and specific primers are important in case.

But, I have problem with only one gene primers, for rest all the primers i don't see any mess, no amplification in negative control, no same ct value or melting curve. everything is perfect. then why only this primers are giving false results?

Please explain on this if u can. thanks

What, exactly, is your 'negative control'?

Is it a cDNA reaction minus the reverse transcriptase, a cDNA reaction minus the RNA, a qPCR reaction without any added cDNA, or is it something else?

Qpcr reaction without cDNA. And normal pcr reaction without any template DNA.

Amplification in a negative control containing no cDNA (end ergo no RNA or gDNA) may indicate either contamination of one of the reagents with template (cDNA or gDNA), or that you have a truly profound primer-dimer issue.

You can check your primers for self-pairing and hetero-pairing using IDT's Oligo Analyzer (https://www.idtdna.com/calc/analyzer).

Likewise, you can try to check for contamination by repeating the experiment with new reagents (including the nuclease free water). If the amplification goes away in the negative control with new reagents, you can confirm that the issue was indeed contamination by combining aliquots of the qCPR reaction made with new and old reagents (e.g. make a titration curve). The negative control amplification should decrease in a dose-dependent manner with dilution, down to the limit of detection for the assay/equipment.

Out of curiosity, is the CT value in the negative control the same as the CT value for the experimental samples? Or is it much higher? Are the CT values for the experimental samples in the 20's? Is the CT value for the negative control higher than 30?