I am working with aloe vera RNA and have been using the Trizol method of extraction of RNA. I have recently started treating my RNA with DNase (Himedia) (1U/microlt for 1Microg RNA). However, I used the treated RNA for cDNA synthesis, and obtained no amplification of my target gene in subsequent PCR. When I checked my DNase treated RNA on AGE, I saw a clean lane with no RNA bands at all. I tried diluting the DNase to 10 and 50 fold, but still my RNA gets degraded.
On the other hand, to get rid of genomic DNA, does using only Oligo dT primers for cDNA synthesis from total RNA ensure that the genomic DNA is taken care of? Please suggest methods for how I could ensure my RNA is free of genomic DNA contamination.
Please note: The target cDNA that I am working with has no intron when analyzed insilico. Hence it has the same length as the genomic sequence.
Well it is not garenteed that your oligo dt will bind only RNA but this is not a big problem because during RT there is no big amplification step. Problem will arise when you have to do a subsequent PCR. If you have genomic DNA contamination in your RT. It will be amplified. There can be different solutions depending on what you want to do with your cDNA later on. If you want to clone some gene from this cDNA. Then You can distinguish your band on the gel. cDNA band will be smaller because your introns will be spliced out. If you are dealing with intronless gene the you don't even need to bother to make cDNA because your genomic DNA and cDNA will essentially have the same sequence. But if you want to quantify the transcripts. You have to design the primers in a way that they do not bind the DNA. That means you should design the primers at intron exon junctions. These primers will only bind your cDNA and you do not need to worry about your DNA contamination. But if you are dealing with intronless genes for their transcript quantification. In that case you have to get rid of your genomic DNA. Depending on your budget there are different kits which exist. Try to use some RNase out (RNase inhibitor) during your RT. I hope it helps
Hi Sharon, did you check your RNA (just after isolation, without DNAse treatment) on PAGE/Agarose Gel Electrophoresis ? Probably, there is problem with the RNA isolation or degradation happening because of RNAse contamination. DNAse should not cleave RNA, so i would suggest checking RNA integrity before DNAse treatment, to make sure your RNA isolation working fine.
Oligo dT primers usually binds to mRNA, but, genomic DNA also contains some polyA streches. Therotically, if oligodT primers find these kind of genomic streches, possibly it can bind. But, anyway, during PCR reaction, you are providing two primers that are specific.
Whats the downstream application you are going to do ? Real Time PCR or Cloning ?
I don't know if this has anything to do with your case, but besides checking for RNA integrity before adding the DNase as stated by the colleagues above, did you try cleaning your samples for the possible presence of polysaccharides?
I remember using Trizol method once to get mRNA from an animal that had high contents of polysaccharide, and whenever I tried to perform PCR, no amplification occurred. Then I read some steps on trizol protocol that mentioned the use of a high salt solution to purify the samples somewhere along the washing steps. If you haven't done so, I think it's something you could try. It worked for me.
Since your genes have no introns, I think the best way out is to get rid of the gDNA to avoid false results regarding the expression. Not sure if Oligo-dT primers alone would do the work. But, maybe you could design the primers to anneal right at the junction between the poly(A) tail and the end of the target gene sequence (if it is a known one). Maybe it would help you to selectively amplify only the gene transcripts instead of the genomic material. Well, at least in theory :)
Increase the length of oligo dT primer and also use the gene specific primer, you well get the amplification from your desired mRNA only not from genomic DNA.
Hi everyone,
I have been isolating RNA for past few months, but never once have I faced RNA degradation problem. I am guessing the contamination is from DNase enzyme that I used. But thank you al for your valuable suggestions. I will try the above. It definitely should help. Thanks
Hi, Sharon
I also work with RNA, and I always check the RNA by AGE twice, after the extraction and after DNase treatment. As you said, in every step, RNA can be degraded and this would make RT/PCR very difficult. Be careful with degradation.
Also, did you design the primers? and if you are working without introns: did you check first if your primers amplified with genomic DNA only? Because if the cDNA you are working with is the same gDNA sequence, and the primers do not amplify with gDNA, I doubt they will amplify the target cDNA.
Hope this help.
Best regards,
to check for DNA background, you could do the PCR reaction on your RNA sample without cDNA synthesis. This should give no (background) band. Do you at least have a positive control which works with these primers?
good luck!
Is this gene house-keeping gene (expressed all the time)? Otherwise, you need to make sure that the gene is still expressing (producing mRNA) while you are isolating the total RNA. Some genes only express at a specific plant development stages.
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