I have obtained 3 inter-related isoforms of lectins (~115 amino acids) with >80% of identity at amino acid level. Alignment shows only two regions of ORFs are sequence specific. To design qPCR oligos, shall I go with 3’ UTRs or those two specific regions? Is there any disadvantage in designing oligo in 3’ UTR regions?
I think specificity of isoforms may be less for 3' UTRs in comparision to sequence specific regions (ORFs). Otherwise for these two reactions qRT-PCR should not be very different. Best will be to establish a plateform.
The 3' UTR is generally used because it is the best region to find sequence variation between closely related isolates. So if your desire is to distinguish between the isolates then use the 3' UTR. However, the 2 regions that are sequence specific would work just as well. If you are using a poly T primer to make your cDNA then the 3' UTR will be represented more frequently then something towards the 5' end. It is best to use random primers to ensure that the cDNA templates are reasonably representative of the entire transcript to avoid bias.
Thank you for your suggestion. In fact, I already have ordered the oligos spanning within ORF. I will be checking their specificity based on dissociation curves. If there is any indication for non-spcific amplification, then as you suggested, I will design new oligos in UTRs.
I performed the qPCR with the oligos falling within ORF (but targeted to a region that significantly vary among 3 lectins. Inspection of dissociation curves indicate that oligos are specifically amplifying corresponding amplicons (See the attachment). Meanwhile, I am progressing the cloning to ensure whether the amplicons are expected or false positives, by sequencing. What else could I do to ensure that I am in the right way towards proper results?
RNA quality and quantity assessment, efficiency of cDNA synthesis and PCR controls will tell the whole story. You must monitor the whole story.
Sunil Pal!, I have performed those steps earlier. In fact, my doubt is regarding any precautions through which I might overcome the amplification of non-specific isoforms when I use a particular pair of oligo. What are the other potential perspectives to proof that I have amplified a fragment which I targeted, other than dissociation curve analysis and sequencing of amplicon?
In my first phase of cloning and sequencing, I found that the oligos I designed were specific only for two lectins; while the third lectin failed to provide a specific amplicon as per sequencing results. Based on Richard McQualter's idea, I designed oligos, in the 3' UTR region for the third gene, amplified and cloned. The sequence result recently obtained, indicated that it is 100% specific for the third gene. Well. Now, I am ready to perform qPCR assays with an objective of comparative profiling of three isoforms. Thanks for your valuable comments.
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