I extracted the RNA and prepared cDNA using the reverse primer of OsActin instead of Random hexamerase and did a PCR for the same, but to my surprise i found two bands of different size of two as expected and other just below in the agarose gel. it is very clear band and nothing like non specific binding or primer dimer, so please can someone suggest me the reasons for it?
Hi Amit
as all genes, housekeeping genes can have several transcripts.
probably you got 2 of them with the primer you designed.
check at a database, NCBI, Ensembl or UCSC to see where you primer is located and see if alternative transcripts are possible.
fred
Hi Amit
as all genes, housekeeping genes can have several transcripts.
probably you got 2 of them with the primer you designed.
check at a database, NCBI, Ensembl or UCSC to see where you primer is located and see if alternative transcripts are possible.
fred
Dear Amit
- I agree with Frederic: I would also add that if you want pan specific amplification in terms of transcripts, make sure you chose a 3' exon (within 2kb of Poly A tail) that is common to all transcripts
- In addtion, check Enseml as well as GenBank for putative transcript structure: I will only design primers to transcripts that both GenBank and Ensembl agree on
- Finally, there is another possibility: If you are performing reverse transcription with an isoform specific primer to increase sensitivity (as opposed to Random Hexamers) you increase the likelyhood of non specific amplifications; This is why I tend not to do amplify as you have suggested; If I know my transscript s low copy number which is a reason for doing what you have done then I tend to amplify all of my cDNA made with random hexamers and then fish out a specific transcript by PCR (with sequence specific primers). If you would like the post cDNA amplification protocol I can send to you
- Another way would be to make cDNA with random hexamers; perform primary PCR with one set of primers and then perform secondary hemi nested PCR with the same reverse primer and an internal forward primer using a 1:100 dilution of the primary PCR reaction
- But for specificity I would always start with random primers and not dive straight in with sequence specific primers
Thank You so much Laurence Stuart Dawkins-Hall
Please it would very helpful for me if you can share the cDNA Amplification protocol.
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