I did cDNA synthesis after RNA extraction including a negative control containing RNA and water with no RT. I used all my samples for regular PCR While I included a negative control containing mastermix, polymerase and primers but no cDNA. I didn't get any bands for PCR negative control but I got bands for RNA negative control ( explained above) which wasn't expected. I am wondering what is the reason.
RNA was extracted by hot SDS lysis protocol using phenol/ chloroform. Then samples were treated with DNase to remove DNA. The cDNA synthesis was done afterward which resulted in average 1400 ng/ul of cDNA in my test samples and about 30 ng/ul in my negative controls. PCR was conducted after using all these tubes plus PCR negative controls.
To be sure that I understand you right: Your no RT control in the cDNA synthesis gives you bands when you use it in PCR? At which size do this bands appear? Does you PCR negative control contain primers? Can you show a picture of the gel?
What do you mean by RNA negative control. Your objective, I presume, is to measure the level of expression of a specific mRNA In your total RNA preparation. If you get a band with your RNA preparation (your RNA control) it means that your RNA preparation is contaminated with DNA. You have to DNAse 1 your RNA preparation. Therefore you will be able to see if your mRNA has been actively synthesized.
Tell me how you prepare your total RNA et from what kind of cells?
RNA was extracted by hot SDS lysis protocol using phenol/ chloroform. Then samples were treated with DNase to remove DNA. The cDNA synthesis was done afterward which resulted in average 1400 ng/ul of cDNA in my test samples and about 30 ng/ul in my negative controls. PCR was conducted afterward using all these tubes plus PCR negative controls.
Ladder / Gap / PCR Negative control1 / Test sample1 / test sample 2 / PCR negative control 2. / RNA negative control 1. / RNA negative control 2
Hi Homa
Your bands in nagative samples is indicative of some sort of contamination....I would suggest you repeat your experiment once more carfully.....it may also be due to some mistake....it is possible
bye
Judging from your gel, your PCR is not the problem, since the negative control lanes are clean.. You get some primer dimers, but that doesn't matter too much.
You RNA samples can not be analyzed at the moment, since the band of interest is present in every lane which contains cDNA, also in the negative control. Since you did not use reverse transcriptase in the RNA negative control samples, this is most likely due to genomic DNA contamination.
Do you do a DNAse digest of your prepared total RNA? This would help to get rid of this contamination. You can still treat the samples you have now and then prepare new cDNA.
Then: Your PCR primers: Are you interested in spliced mRNA? If so, you could use primers, which span intron-exon-boundaries, meaning the primer begins in one exon and ends in the next. Amplification happen only on DNA which has no intron inbetween (meaning cDNA derived from mRNA).
Dear Christian,
I have tried DNase both in tube and column. Unfortunately I am still getting bands in RNA negative control. The gene I am working doesn't have intron so I can not use that method. Probably the protocol I am using is not efficient. Can you suggest me any good protocol for RNA extraction that you have tried before and it worked for you?!
@Homa: An intronless gene makes the game harder. I usually used the protocol with any of the Tri reagents available, followed by a DNAse I digest and clean-up on RNAeasy columns.
How do you treat your samples with DNase I?! I'd like to compare your method with mine.
If all other things are controlled, your results are due to DNA contamination of your total RNA preparation. Remember that even a very low amount of DNA in your RNA preparation is sufficient to be amplified by this experimental approach. This is the miracle of PCR.
Sometimes DNAse treatment does not work properly. It depends of the quality of your DNAse, the concentration used, the time of incubation, etc. You should perform a dose response experiments and modify the times of incubation, etc. When I was doing this type of experiments long time ago I often performed two succesive DNAse treatments to get rid of all DNA.
@Homa: We are using the standard protocol from Qiagen for the on-column digest with no differences.
Dear catherine, I also did two DNase treatment unfortunately my RNA was degraded too cause I got no band even in my positive control. Can you please share the protocol you were using?! Thanks
Thanks Christian. I f anything else failed I will try to get these kit.
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