I would like to design primers for RT-qpcr for my gene. I understand that to avoid amplification from potentially gDNA contaminant, primers designed should span across an intron so that we can manipulate extension time to optimize amplification of fragment from cDNA which is smaller than fragment from gDNA due to absence of intron.
My gene consists of:
Exon1 (770bp) - Intron1 (70bp) - Exon2 (590bp) - Intron2 (57bp) - Exon3(20bp)
However, my introns are quite small, can the above concept still be applicable? What is the optimal fragment size to amplify for RT-qpcr? Is 150-250bp ok?
if you designed a forward primer of the last 10 bases of exon1 and the first 10 or more bases ( approximate)of exon 2 this primer would only work on cdna then the reverse can be in exon 2 and this mix should not amplify gdna
Hi Vikki
Paul Rutland is right, another strategy of qPCR primer designing (apart from intron spanning) is to put your your primers on splice junctions. This will help with small introns.
Good luck
You can try ncbi primer design
https://www.ncbi.nlm.nih.gov/tools/primer-blast/
Regards
Hi! I agrre with Nashaat Ghalib Paul Rutland Ali Javadmanesh
1) Design forward or reverse primers at exon1-exon2 or exon2-exon3 junctions, then gDNA will not be amplified.
2) Alternatively, you can do a DNAse treatment in your RNA before reverse transcription. There are several kits with gDNA removal during RNA extraction and reverse transcription.
Try no reverse transcription reactions to see if you have gDNA contamination.
Of course, this aprroach (2) is only possible if you do a two step qPCR protocol, separating reverse transcription and qPCR.
Best,
Jacó
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