I am working on bovine rotavirus in Egypt, i searched and found little number of papers doing RT-PCR in Egypt
What are the parameters that make me choose the correct one?
the RNA nature of the virus makes me worried if i pick old or non working primer
thanks
I have no experience with bovine rotavirus, so I cannot give you any specifics. If I was you, I would order several primer pairs that you in the literature and try them all. The cost of the primers is usually not very high compared to the rest of the consumables, so giving several primer pairs a try at various conditions (annealing temp., primer conc. Taq conc., etc.) might be your best bet. Just be sure that the primer actually target what you are looking for.
If that doesn't work, you could look into designing your own primers. (This is usually also a good way to get your paper cited more often--as others who might use the primers you designed would cite your study.)
By the way, when you say "RT-PCR" do you mean "reverse transcription PCR" or "real time PCR"?
I hope this helps.
Cheers, Oliver
thanks
i meant reverse transcriptase PCR
i found an egyptian paper but i think the PCR work is fake
the gel look so fake
Dear Reham,
I suppose you should choose the most cited, dependable paper to start with.
Abhishek
Dear Reham,
Assalamo-3alikom, how are you & how is your work? have you solved the problem of choosing the correct primers or not yet?
You can go Dr. Reham either with primer designing or choosing the primers from reputable journals such as Journal of Virology, Veterinary Microbiology, JCM, etc.
Good luck
- Web-based high-throughput Primer Design using the following online programs:
*Primer-Blast, link, http://www.ncbi.nlm.nih.gov/tools/primer-blast/
*Primer3plus, link, http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi
*Primer3Web, version 4, link, http://primer3.ut.ee/
*Primer3, link, http://bioinfo.ut.ee/primer3-0.4.0/
- Web-based Checking Primer Design using the following online programs:
*Oligo Calc: Oligonucleotide Properties Calculator, link,
http://biotools.nubic.northwestern.edu/OligoCalc.html
*In silico PCR, link, http://insilico.ehu.es/PCR/
Generally, for me, working in RNA viruses is not easy. there are many aspects for answering for your question. sometimes i used the most cited articles. in another cases i design my own primers. Gooood luck.
It is OK, you can choose the primers you need from papers published in international journals of high impact factors (Thomson reuters), I usually make this, but I am afraid as once, I selected primers that cited in many papers published in reputable journals and I found no honest in PCR conditions and the result was negative for all samples even the control positive. I tried to use gradient PCR to select the most suitable annealing temperatures for the primers which were so far from that cited in the published papers and gave me acceptable results.
So, you can select your primer from a published paper then check it using programs I sent you before.
Yes, i agree with you dr Norhan. sometimes we went with primers published in reputable journals and we noticed that annealing temp. was completely different. For me to figure this issue, i check only 2 annealing temperatures -4 & +4 to the cited one.
OK Dr Hazem, this is very good, but you will be amazed when I tell you that the annealing temperature in the paper was 44 °C and that with gradient PCR was 55 °C. Sometimes there is no honest in this matter.
Yes, 11 degrees difference is too big and i don't know if they did it intentionally or not. By the way i had something like this and i contacted the authors themselves for getting more information, the paper was published in AEM.
thanks sooo much Dr-Norhan , Dr -Mohamed Sultan and Dr-Hazem for the helpful discussion,
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