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mRNA Search for RT-PCR (U to T)


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#1 Shilpagoel

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Posted 23 March 2011 - 08:28 AM

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.

#2 Trof

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Posted 23 March 2011 - 01:03 PM

Take the "mRNA sequence" from your link and feed it to primer design software (like primer3) and just design primers. It works.

RNA can't be sequenced AFAIK, co this probably is a complementary sequence to complementary sequence to original mRNA (so having the same sequence as original mRNA), so it has the same triplets and everything but without any uracil there.

If I'm not mistaken, I see it like this:
- mRNA is first reverse transcribed to cDNA, cDNA is only single stranded and complementary do original mRNA sequence
- only primer complementary to cDNA will amplify (or more precisly create the second strand) in the first cycle, that means it has to have same sequence as mRNA, when you get double stranded cDNA, everything is same as for DNA
- primer design software designs forward primer having the same sequence as target sequence = mRNA, so it's complementary to cDNA which is what you need

Result: It works.

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#3 phucvn

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Posted 23 March 2011 - 09:59 PM

Yes, I agree with Veteran.
I don't know why Databases (GenBank, DDBJ, EMBL) decide to deposit mRNA sequences under DNA nucleotides (ACGT) but not RNA nucleotides (ACGU) that make people confused like this guy. I believe that other people also confuse like him.




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