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Library quantification of Small-RNAseq

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  • Library quantification of Small-RNAseq

    Hi!
    We are doing Small-RNAseq libraries since a little bit (using TruSeq Small RNA prep kit) and we have problem quantifying the libraries accurately. We are using the KAPA quantification kit, but we have very variable read counts on the HiSeq with this technique. We suspect that KAPA quantification kit is not optimal for quantifying this kind of libraries since the standards are longer than the library fragments.
    What quantification technique do you guys use for Small-RNAseq?
    Thanks!

  • #2
    We are using Qubit for quantification for the purpose of pooling the libraries and Kapa quant to quantify the final pooled library; so far it seems to work quite well.

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    • #3
      moving to Sample prep per request!

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      • #4
        Are you mixing your small RNA libraries with other library types for sequencing? My understanding is that smaller products generate clusters more efficiently than larger ones, so maybe a difference in ratio of small RNA to "normal" libraries could explain the variable read counts?

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        • #5
          I've been using a Bioanalyzer for quantification which works well for the most part but sometimes you have to manually adjust the integration for peaks with shoulders.
          For a crude sample just after PCR, wouldn't the Qubit quantify all the double stranded products in your sample and not just your library band of interest?

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          • #6
            From our experience, using Bioanalyzer first and then KAPA. This will give you better results. In addition, after you mix your libraries, repeat Bioanalyzer and KAPA again.

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            • #7
              We just use Bioanalyzer QC. Most of the library will be tRNA. It's more important to know what percentage is miRNA

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              • #8
                Originally posted by luc View Post
                We are using Qubit for quantification for the purpose of pooling the libraries
                Are you pooling your libraries before or after you size select on a gel?

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                • #9
                  Originally posted by EdibleMetal View Post
                  Are you pooling your libraries before or after you size select on a gel?
                  We are pooling after size selection and bioanalyzer QC (of the size selection) according to Qubit measurements -- might be too much.

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                  • #10
                    "We suspect that KAPA quantification kit is not optimal for quantifying this kind of libraries since the standards are longer than the library fragments."

                    You can do an adjustment of the length by this formula:
                    Final quantity= nM quantity*(standard length/insert size)

                    Hope this helps

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                    • #11
                      I have a very naive question. What do you use to quantify RNA if your input is a small RNA purified fraction ? Thanks

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                      • #12
                        Originally posted by NextGenSeq View Post
                        We just use Bioanalyzer QC. Most of the library will be tRNA. It's more important to know what percentage is miRNA
                        Hi NextGenSeq,
                        I know I'm asking a question about a nearly nine-month-old post, but anyway:

                        My understanding of the TruSeq small RNA kit was that nearly all of the actual library molecules should be miRNA or piwiRNA derived because of the ligation/size-selection used. The ligation step actually involves using T4RNAligase1 to ligate oligos directly to these RNAs. For a ligation to occur the RNA ligated-to must have a 5'-phosphate and 3'-hydroxyl. Most RNA will have a 3'-phosphate/5'-hydroxyl and won't participate. tRNA would be one of the exceptions, I guess. But there is a size selection using an acrylamide gel that should get rid of the tRNA-derived cDNAs, right?

                        --
                        Phillip

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                        • #13
                          Originally posted by pmiguel View Post
                          Hi NextGenSeq,
                          I know I'm asking a question about a nearly nine-month-old post, but anyway:

                          My understanding of the TruSeq small RNA kit was that nearly all of the actual library molecules should be miRNA or piwiRNA derived because of the ligation/size-selection used. The ligation step actually involves using T4RNAligase1 to ligate oligos directly to these RNAs. For a ligation to occur the RNA ligated-to must have a 5'-phosphate and 3'-hydroxyl. Most RNA will have a 3'-phosphate/5'-hydroxyl and won't participate. tRNA would be one of the exceptions, I guess. But there is a size selection using an acrylamide gel that should get rid of the tRNA-derived cDNAs, right?

                          --
                          Phillip
                          Hi Phillip,
                          yes the protocol include a gel-purification whereby you cut out the band including the small RNA fraction+adapters (150nt) and discarding the 200 nt band with tRNA plus adapters

                          Comment


                          • #14
                            Originally posted by pmiguel View Post
                            Hi NextGenSeq,
                            I know I'm asking a question about a nearly nine-month-old post, but anyway:

                            My understanding of the TruSeq small RNA kit was that nearly all of the actual library molecules should be miRNA or piwiRNA derived because of the ligation/size-selection used. The ligation step actually involves using T4RNAligase1 to ligate oligos directly to these RNAs. For a ligation to occur the RNA ligated-to must have a 5'-phosphate and 3'-hydroxyl. Most RNA will have a 3'-phosphate/5'-hydroxyl and won't participate. tRNA would be one of the exceptions, I guess. But there is a size selection using an acrylamide gel that should get rid of the tRNA-derived cDNAs, right?

                            --
                            Phillip

                            After PCR you typically end up with a decent amount of higher molecular weight products that represent tRNAs and other cellular RNAs, and PAGE selection does allow you to exclude these from your final library. But the real reason PAGE purification is typically necessary is that adapter-dimer product is only ~20 bp smaller than miRNA product, so size selection with Ampure XP is not feasible.
                            Last edited by kerplunk412; 10-09-2015, 03:30 PM.

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                            • #15
                              Originally posted by robertami View Post
                              I have a very naive question. What do you use to quantify RNA if your input is a small RNA purified fraction ? Thanks
                              I use small RNA Chip because one can check the success of small RNA isolation as well. It should be possible to use PicoGreen reagent as well, but that will not give indication of size distribution.

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