I did a PCR for cDNA from 3 different tissues, 2 of which do not express my gene of interest. While only the predicted tissue showed the band for my gene of interest, the negative controls for each one of these showed a band larger than my gene of interest. The negative control is RNA which has not been reverse transcribed. Even though negative controls are used for checking contamination, can I still make the conclusion that my PCR worked? What would the larger size of these bands suggest? Thank you
If you are certain you DNase treated all of your RNAs with 100% effectiveness, there should be no gDNA contamination unless it floated in from the room you are working in. Have you BLASTed your primers to be sure they aren't complementary to a gDNA sequence in the negative tissues? If the 3 tissues you are testing are from the same organism, then gDNA +intron is the longer product you have amplified (if you are working in a eukaryotic system that is). Without further detail - these are all guesses. What organism? What tissues? Did you design primers to span an exon-exon junction?, etc.
If you are certain you DNase treated all of your RNAs with 100% effectiveness, there should be no gDNA contamination unless it floated in from the room you are working in. Have you BLASTed your primers to be sure they aren't complementary to a gDNA sequence in the negative tissues? If the 3 tissues you are testing are from the same organism, then gDNA +intron is the longer product you have amplified (if you are working in a eukaryotic system that is). Without further detail - these are all guesses. What organism? What tissues? Did you design primers to span an exon-exon junction?, etc.
Since you did not see these bands in your samples, but only in your negative controls , I would say you have gotten a contamination by DNA genomic (if you have a very big band) in somehow and only for the negative controls, which is quite strange. Contamination by gDNA was more credible if that big band was present in all samples.
I would suggest to repeat the PCR again and using new reagents to see if you will get the same results. There is also the possibility you have forgotten to put something (for example the template the RNA or cDNA) in those samples where you did not get any bands.
BTW, The contaminating genomic DNA in RNA samples can significantly affect the Ct value of subsequent qPCR reactions, especially for genes with low transcription levels.
Importantly, after DNase, you need to verify that the RNA is devoid of DNA. Just run a qPCR with DNA-specific primers (i.e. that do work on DNA) on RNA. As RNA is no template for PCR, any signal you get is from contaminating DNA. This is an example. or use your primers on your RNAs and check that they are cleaned from higher bands.
Hi Kunal,
Do not worry too much. There is a mean to escape from unwanted DNA bands in negative controls.
This is the Agilent product named Perfect Metch PCR Enhancer. Just add one microliter to your PCR mix and you will get VERY specific amplifcationsresulting int he loss of almost ALL non-specific amplifications.
Just try it in your case!
Best regards
Philippe
The bands are not primer dimers if large and therefore must have come from genomic DNA contamination, or non-specific binding to another cDNA. Non-specific priming can sometimes happen when your primers aren't finding a specific target to amplify (negative controls) and there is too much cDNA in the sample. False priming happens at higher frequency when the annealing temperature is too cold. False priming can also happen if you have active polymerase in a tube held at room temperature as you set up the reaction (perhaps you are using hot start polymerase, which stops this problem).
Recommend re-assessing your PCR conditions and methods, and/or remaking the RNA with a more thorough DNase treatment.
You did not specify if you are using eukaryotic RNA or prokaryotic. Or if you quantified the RNA prior to PCR. There is likely to be more copies of mRNA in your negative control than copies of the cDNA in your test (presuming you didn't use gene specific primers to create your cDNA). The product in your negative control can be explained by amplification of the larger quantities of mRNA ( unspliced) in your control.
How do you know the two tissue are negative for gene expression?
Hi.......
It may be due to genomic DNA contamination or alternate splicing of gene if any or may be contaminatin depends on first two.
HI, I agree that it is probably contamination with gDNA. It sounds a very logical solution to the larger bands.
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