Basically i am running .
Edited by hianghao, 14 October 2010 - 01:58 AM.
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How to deal with truncated mRNA or mRNA that has secondary structure
Started by hianghao, Oct 13 2010 05:27 PM
2 replies to this topic
#1
hianghao
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Posted 13 October 2010 - 05:27 PM
I got band of unexpected size (100bp) from 5' RACE (using SMARTer RACE kit from Clontech) using cDNA as template, which is suppose to be 1kbp. It seems that it might due to high secondary structure in mRNA or the mRNA is truncated. I don't understand what is truncated and secondary structure. I tried using 1st strand cDNAs synthesized by oligo dT and random primer and the results were the same. I've also tried 3 different primers and still, same problem.
Basically i am running .
Basically i am running .
#2
Trof
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Posted 14 October 2010 - 06:05 AM
Unresolved secondary structure in mRNA would IMHO prevent amplification at all, not create a smaller product.
I don't know details of your kit, but in my kit 5' RACE oligo is ligated only to a intact GTP head (if I remember it right) so fragmented mRNA wouldn't amplify.
So if your kit wirks the same and all steps were done right, there still remains possibility, that apart from your expected 1kb transcript there is another one, which is smaller and so your PCR is correct.
I don't know details of your kit, but in my kit 5' RACE oligo is ligated only to a intact GTP head (if I remember it right) so fragmented mRNA wouldn't amplify.
So if your kit wirks the same and all steps were done right, there still remains possibility, that apart from your expected 1kb transcript there is another one, which is smaller and so your PCR is correct.
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#3
hianghao
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Posted 14 October 2010 - 03:33 PM
Trof, on 14 October 2010 - 06:05 AM, said:
Unresolved secondary structure in mRNA would IMHO prevent amplification at all, not create a smaller product.
I don't know details of your kit, but in my kit 5' RACE oligo is ligated only to a intact GTP head (if I remember it right) so fragmented mRNA wouldn't amplify.
So if your kit wirks the same and all steps were done right, there still remains possibility, that apart from your expected 1kb transcript there is another one, which is smaller and so your PCR is correct.
I don't know details of your kit, but in my kit 5' RACE oligo is ligated only to a intact GTP head (if I remember it right) so fragmented mRNA wouldn't amplify.
So if your kit wirks the same and all steps were done right, there still remains possibility, that apart from your expected 1kb transcript there is another one, which is smaller and so your PCR is correct.
Thanks for replying. But i really doubt that the smaller transcript exist. Are you using Clontech kit as well?
Edited by hianghao, 14 October 2010 - 03:52 PM.
