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  • Isolation of mRNA

    Hi everyone, just a quick question.

    I am using a library protocol that uses a magnetic poly(A) isolation of mRNA, followed by a random hexamer based first strand cDNA synthesis step. The poly(A) kit is expensive and doesn't work well on smaller sample sizes, which I am using. My question is, why don't I just use a oligo(d)T based primer instead of a hexamer primer in my first strand synthesis and skip the poly(A) magnetic isolation. Why doesn't everyone do this instead of a magnetic poly(A) isolation step... do I expect more yield losses or rRNA contamination, or a substantially greater bias towards the poly(A) tail?

    Thanks in advance,
    Mike

  • #2
    It depends on the type of library that you are preparing and experiment aim. If you use oligo(d)T, your cDNA and resulting library will have 3’ bias as you have mentioned and you will not have much control on fragment length. For optimal results and analysis NGS libraries require certain insert size. I do not think that it will affect rRNA contamination level.

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    • #3
      Hi Mike,
      At some point a full length mRNA needs to be broken into smaller fragments for Illumina or Ion sequencing. The options are to do this fragmentation on the RNA or on the double-stranded DNA, and it is typically faster, easier, and cheaper to do a chemical/heat (or enzymatic for Ion RNA-Seq) fragmentation of RNA rather than an acoustic or enzymatic shearing of double stranded DNA. Another advantage of fragmenting RNA and then priming with random hexamers is that the degree of 3' bias should not correlate with the length of the mRNA, which it typically will in oligo (d)T primed samples. Priming with random hexamers also provides more flexibility in working with different samples types, for example non-polyadenylated mRNAs from prokaryotes or partially degraded RNA samples.

      Also, you did not mention what poly(A) isolation kit you are using, but there are probably less expensive options out there. For example my employer, Bioo Scientific, offers very cost-effective poly(A) beads.

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      • #4
        Thank you for the replies. They are much appreciated. Those kits are quite affordable for poly(A) isolation, but I am working with samples in about the 100-200 ng range, which is below the input for these kits. The poly(A) kits for bead isolation of small samples tend to be very expensive and tend to lose a lot of yield.

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