mRNA Search for RT-PCR (U to T)

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    • #14714
      shilpagoel
      Participant

      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.

    • #104151
      JackBean
      Participant

      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 😉

    • #104153
      angel92
      Participant

      how’s this possible that in mRNA sequence there is no uracil 😛

    • #104164
      shilpagoel
      Participant

      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

    • #104165
      SLKilbride
      Participant

      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.

    • #104166
      shilpagoel
      Participant

      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?

    • #104172
      SLKilbride
      Participant

      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?

    • #114450
      microniranjan
      Participant

      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?

    • #115000
      JackBean
      Participant

      Because first you synthesise 1st strand and then use the primers to amplify your gene.

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