mRNA Search for RT-PCR (U to T)

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shilpagoel
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mRNA Search for RT-PCR (U to T)

Post by shilpagoel » Wed Mar 23, 2011 4:35 pm

Hi All,
This probably will be rated as the most silly question. I am trying to design primers for RT-PCR. I am assuming here that I will have to find an mRNA sequence of interest and then design complementary primers for the mRNA sequence.
When I search for mRNA sequence I get sequences that do not have Uracil (u) but have thymine (T) in the sequence. Should the RNA not have (u) substituted for (t)?
For the enzyme enos I get a http://www.ncbi.nlm....ore/NM_008713.4
Or for pdgfr beta I get
http://www.ncbi.nlm..../NM_001146268.1
I am new to this but will be really grateful if someone knowledgeable can help me with this.

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JackBean
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Post by JackBean » Thu Mar 24, 2011 8:06 am

your links are broken

it doesn't matter that much. Both cDNA and mRNA have the same sequence, except the T/U, so you can design primers for either and it should work ;)
Even, usually the RT-PCR is done with oligo-dT, so that you get 1st strand cDNA, which is more stable than RNA and then you can easily perform "regular" PCR ;)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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Post by angel92 » Thu Mar 24, 2011 4:18 pm

how's this possible that in mRNA sequence there is no uracil :P

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Post by shilpagoel » Fri Mar 25, 2011 3:05 pm

Sorry for the broken link,
The correct link is

For eNOS mRNA

http://www.ncbi.nlm.nih.gov/nuccore/NM_008713

For PDGFRbeta mRNA

http://www.ncbi.nlm.nih.gov/nuccore/NM_001146268.1

1) Can you kindly please post a link to any mRNA sequence from ncbi website form nucleotite ,genbank etc which has U and not T?
2) Can you please confirm that the sequence in the above link can be used for RT PCR primer design?
Thanks
Shilpa

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Re: mRNA Search for RT-PCR (U to T)

Post by SLKilbride » Fri Mar 25, 2011 9:34 pm

Hello shilpagoel, I believe what you are referring to is the DNA sequence at the bottom of the pages. This the DNA sequence that encodes the mRNA, not the mRNA code itself.

shilpagoel
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Post by shilpagoel » Fri Mar 25, 2011 9:55 pm

Hi SLKbride ,
Thanks for the reply. If this is a DNA sequence then I would not be able to design primers from it. Correct?
would you be able to pase a link for a mRNA sqequence, any protein will be all right?

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Post by SLKilbride » Sat Mar 26, 2011 12:28 pm

Hello shilpagoel, the DNA sequence is cDNA and can be used to design primers for the mRNA sequence that you are after. The introns have already been spliced out, so whats shown on the NCBI is the final transcript. Just copy and paste the sequence into Word and use the 'Find and Replace' function to change thymine (t) to uracil (u). Do you know how long you want your primers to be?

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Re: mRNA Search for RT-PCR (U to T)

Post by microniranjan » Sat Oct 05, 2013 9:58 am

If we design primers to one strand out of two strands of cDNA then there is a chance that one of the primer will not be complementary to mRNA and it will not bind to mRNA.How can we synthesize cDNA from that mRNA?

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Post by JackBean » Fri Jan 24, 2014 12:58 pm

Because first you synthesise 1st strand and then use the primers to amplify your gene.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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