I have extracted rna from insect samples. And designed primers for PCR but none of them are working. I have checked the RNA quality it is about 1.7, but actin primers are working well. Can anybody give me suggestion to proceed my work. Thanks in advance.
Did you transform your RNA into cDNA? Which genes are you testing using PCR?
Make sure that your genes are expressed in the tissues organs that you examine. Your genes may are not highly expressed in the certain conditions. Extract total RNA from tissues that yours genes are expressed find a previous published work. Check your primers and make sure that are designed against the exons it is essential to use est libraries and not genomic.
I second making sure your gene is expressed in the tissues. If the data is not available, do a qPCR on RNA (cDNA) from tissues you know express your gene of interest.
Also, you should start with a primer efficiency test if you've designed these primers yourself.
The actin control primer is probably well tested - if you haven't tested your primers, you don't know if your problem is the template or the primers.
I remember that the red pigment of fruit fly eyes was thought to interfere with enzymatic reactions. Run a spectrophotmetric scan to assess quality.
First of all thanks for your valuable suggestions. Almost i tried all possible way like cDNA conversion using RT PCR, different annealing temperature and different Primers(degenerated Primers) sets also But nothing working. Other Gene primers like actin and 18s primers are working well.
Sounds like your GOI primers are bad. Have a colleague help you download the sequence into an IDT or NIH "primer finder" program. As long as your reference sequence is correct, these programs will find primer pairs that will work!
Are you sure that your insect genus/species have this gene? Perhaps it is lacking or is another isoform. Another possibility is that the gene is silenced. Select primers that can discriminate between mRNA and gDNA. Please let us know your GOI.
The primers could still be OK. If you're only testing them on tissues where the GOI is not expressed, it is not surprising that you get bad results.
How much input cDNA are you using?
As I suggested earlier, it is very hard to optimize the primers on a template with low/no detectable expression of the gene. In my case, a overexpression construct solved the problem. :) Prior to this all my 5 primersets failed for my particular GOI!
Yes Michael sir. my gene of interest must be there in all insects, because Chitin synthase is my GOI. And i had chosen at specific stage where this gene will express more. And please tell me how to discriminate between mRNA and gDNA primers.
Find an intron of about 200 bp if you can. Design primers for the 5 prime exon and the 3 prime exon about 200 bp distal to each. PCR of DNA vs cDNA will be clear.
Okay sir thanks for your suggestion. I will do this first and let you know.
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