I extracted the total RNA from prostate tissue of rat and convert it to cDNA. Then, I run a PCR with specific F and R primers for p53. I looking for 149 bp band but I found only one band with 210-220 bp.
Can anyone discuses about it?
Please also express the ways to overcome this problem.
Thanks
One way to overcome this is to make sure that all your PCR components are free of any contamination. So do put a No template control (NTC) and a positive control in your set of experiment to make sure that everything is fine. Also make sure of your primers (DESIGN and Annealing temperature before PCR), which are supposed to give you product size 149bp.
What electrophoresis buffer are you using?
Some buffers like Sodium Borate and TAE, can distort the results depending on your voltage setting, loading buffer and buffer of your ladder.
please see link below and good luck
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0011318
Did you try to run a non reversed RNA in addition to the cDNA and No-template as a control that you do not amplify by chance genomic DNA? Also you should BLAST both primers to be sure they do not hybridize to other sequences accept the gene of interest. Another thing is how you RT your RNA into cDNA as using only poly dT or only ranom primers give also lower quality of results. Better to use the mix.
Thanks all dear
I used random hexamer when I tried to convert RNA to cDNA. I also checked the primers in NCBI for its specificity. Finally, I used TAE buffer for gel preparation.
I also run a temperature gradient PCR to find the best annealing temperature.
I want to know that "what is this 210-220 bp band despite of using very specific designed primer for p53?"
May be your primers are in different exsons and the genomic DNA has a intron ca. 100 bp (between Exon 5 and 6 or 8 and 9). 210 bp-Fragment is the genomic DNA, but not the cDNA amplificate and your cDNA-Synthesis is not effective. Try to amplificate your samples bevor the cDNA Synthesis to confirm this.
Exactly! This was my suggestion at the first place run your RNA instead of cDNA to see if you get a band, as than it is the gDNA you amplify.
I was run the extracted RNA before cDNA synthesis on 2% agarose gel and use of 1 Kbp ladder.
However, we could not found any bands (neither 18S rRNA, nor 28 srRNA)
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