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Northern blot analysis


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#1 gayle

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Posted 12 February 2005 - 06:23 PM

Hi

I am doing northern blot analysis to detect alternatively spliced form of RNA.
I have bought random primer labelling kit from ambion
If convert my RNA to cDNA and run a gel and then hybridize it with a cDNA probe, will i be able to detect alternatively spliced form of RNA.

Thanks,
gayle

#2 Great White Northern

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Posted 05 March 2005 - 08:57 AM

I may be wrong about this, but I think your amount of cDNA will be very low. Also, due to varying efficiencies of 1st strand sythesis, I think you will see signals a various sizes, even if there is no alternative splicing. I think your best best is 3'RACE or RT-PCR using primers specific to the suspected exons (if you know them). Then, you can probe the PCR products for identity.

#3 gayle

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Posted 07 March 2005 - 10:49 AM

I may be wrong about this, but I think your amount of cDNA will be very low.  Also, due to varying efficiencies of 1st strand sythesis, I think you will see signals a various sizes, even if there is no alternative splicing.  I think your best best is 3'RACE or RT-PCR using primers specific to the suspected exons (if you know them).  Then, you can probe the PCR products for identity.

hi

I have amplified my cDNA by PCR so the concentration of cDNA should be ok
Also, I had done RT-PCR but could not detect any alternatively spliced forms. So I thought that Northern blot using cDNA would be better to detect spliced forms. Is northern blot better than RT-PCR?

sincerely,
Gayle

#4 Great White Northern

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Posted 07 March 2005 - 12:03 PM

Hi Gayle
Ideally, the northern blot using total or polyA+ RNA is the way to go, unless you think that the two splice variants are of roughly the same size.
I don't know anything about your gene or organism, but have you tried to BLAST your sequence (if you know it) against the EST databank? You may get hits against more than one splice variant.
You could also try S1 nuclease or primer extension analysis if this is really giving you a hard time.




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