In this issue I have 2 main questions!
1) DNase treatment
I used mouse-muscle cells as sample.After isolating RNA using the protocol supplied, ISOGEN, I measured the RNA and got an A260/A280 ratio 1.93-1.98 (1.7~2.3 mg) using Nanodrop.
DNase treatment was done by Recombinant DNAse I (RNase-free) and inactivate DNaseI via phenol/chloroform extraction according to manufacturer's protocol. Then, confirm the genomic DNA is removed completely by running electrophoresis. The results came out negative more than I expected. Please have a look to the agarose gel picture (1.5%gel) attached.
In my view,can there be genomic DNA contamination in an RNA sample?
2) select primers spanning exon-exon for targeted genes
I have designed primers spanning exon-exon junction for RT-PCR using Primer3 plus and Primer blast software. However, I still got the multiple bands on agarose gel. PCR condition, I set 50 cycles and the concentration of primers were 100 µM/gene/sample.
I would be very grateful if you could help me in this matter. Any suggestions are welcome.
Thank you in advance.
Q1. I suspect yes, to confirm that we can do a PCR test using universal primers for the species of study (using DNAse treated cleaned up RNA sample as the template + a positive control which is DNA template + a negative control : use water that will be used for RT-PCR instead of DNA) and subsequent gel analysis.
Q2. I suspect primer dimer is present (~100bp marker band), non-specific amplification is obvious. To prevent those, try to reduce primer concentration to 0.3µM. This amount worked for our lab. Perform thermal gradient for determination of optimum Tm for primers (use DNA template of your species of study) (this step may recommend a Tm to be used that avoid primer dimer).
really you're adopting too high primer concentration... it's extremely easy to get aspecific products.
it seems that you actually have DNA contamination.
Thank you all for your suggestion! I will decrease the primer concentration..
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