Hi everyone,
I have been facing with a very big challenge to design some primers for qPCR. My problem is that I need to design some primers to test the expression of some stressed genes in different experimental conditions. Unfortunately, my working species is not yet full-genome sequenced, actually, what I have in my hand so far is just some sequences from a transcriptome database. I did try to test some primers from other papers (with a very good alignment with the sequences that I have) but didn't success.
Now, I want to design some primers by using that sequences but I have no idea how to identify the exon exon boundary within the sequences. Because, I could easily use Primer3 or IDT web tool to design primers but still don't know if these primers span the exon-exon boundary.
So, the main question is how to define the exon exon boundary with a sequence without knowing the whole genome sequences.
It has costed me over 3 month dealing with this question without successful. So, If someone could give me some idea to solve the problem, I would be greatly thankful
Hung.
Hu Huang,
Splign would be helpful for your case:
https://www.ncbi.nlm.nih.gov/sutils/splign/splign.cgi
Also, keep it in your mind that designing the primers which span the exon-exon junction, is required when you do not treat your RNA samples with DNase. But, if you treat the extracted RNA with DNase properly, then you can design primers wherever in your known sequences. When there is no DNA, the primers will anneal to cDNA specifically and your results would be accurate and reliable.
Hi Hossein,
Thank you so much, that is actually a very great idea to use DNase to treat RNA samples before making cDNA. Did you use DNase to treat your samples before? just in case, you did Could you give me the name of the DNase that you used before by chance?
Many thanks,
Hung.
Hi again Huang,
I usually use PureLink™ RNA Mini Kit for RNA extraction. You can order PureLink™ DNase Set from the same company to merge RNA extraction and DNA removal.
Everything is well described in the protocol of kit, which is attached here. Read it carefully to make sure that it is useful for your case.
Regards,
Hossein
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