difference between primer extension and RACE - (Nov/08/2005 )

hellp everyone
i am a new in this field .recently i want to identify a the potential transcription start sites.
in my eye, both RACE and primer extension are the function to identify the transcription start sites .is this idea right?

thanks a million for any suggestions!

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nobody can explain this question for me ?

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Yes, that's correct. Either will work, as will things like S1 nuclease protection assays. RACE has the advantage that it doesn't use radioactive labeling, which, at least for us, is a deciding factor.

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HI
I am working with these techniques for a while now
techniques to be used for determination of transcription start site depends on a lots of factors:
1) how much is already known abt the structure if the gene
2) presence of transcript variants

The first thing would be to do a northern blot to see what size ur RNA is
then you woudl do a 5'-RACE PCR to determine the full length cDNA clone ..Clone the PCR procduct and sequence it
using a primer close to the 5'- end of this sequence to do a primer extension and confirm the result
alternatively RNAse protection assay is used which is a much better characterized technique
u have to design a antisense probe of 100-200bp that encompasses the transcription start site
s1 nuclease might be used too
none of these techniques is easy and it take time to optimize these
and appropriate controls shud be thought of

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Can't you use RT-PCR with a variety of different primers as well? You can determine if the full-length transcript is expressed at all. With qPCR you might even be able to compare the amount of truncated product to full-length transcript to see if there is a difference.

RLM-RACE seems like a very expensive approach to the problem...it is also possible that there are multiple start sites as genes are not always universally transcribed in one way, so if you do the RLM-RACE method, you will have to sequence quite a few clones to make sure that the transcriptome population is uniform.

I think the Northern Blot is the slam dunk methodology for this but qRT-PCR is cheaper and less tedious. If you are publishing these results, do the Northern Blot.

-Matt

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Thanks for your suggestions..
I have certain more questions
if the transcripts differ only by 50 or 100 bp would a northern blot using a 1.5 or 2% gel to fractionate your RNA be able to differentiate between these alternative forms..I ask this because I have literally no experience with northern blot and I am just starting to desgn my probe

the other question is about the RT-PCR ...one might not have the full length cDNA with the oligo-dT primer and so if one doesnt see a band on RT-PCR it doesnt mean that the transcript is not there.
does it? One would have ot use a gene-specific primer more closer to the 5'-end to make the cDNA in the RT step
According to me it takes much more time to set up Real time quantitative PCR than northern blot
Please give me your suggestions and contradictions..

thanks a bunch in advance

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Yes, you should be able to visualize a 50 -100 bp difference on a 2% gel: I know I can.

For your concern regarding lack of full-length cDNA transcript:Why not use random hexamer primers then if that is a concern? It has never been a problem with me. If there is absolutely any full-length cDNA in there, your primer will find it. Even with rare transcripts amplification should not be a problem.

How is RT-PCR more difficult to setup than Northern Blot? I mean, you have to design and label the probes with Northerns and take more precautions with RNA degradation...doesn't blotting take three days with the washes? This is all just in my limited experience, of course...some Northern experts out there might disagree.

-Matt

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