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Why aren't my SPRI beads doing a size-selection?

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  • Why aren't my SPRI beads doing a size-selection?

    I'm thinking about using SPRI beads for size-selections in my library prep, but I thought I'd validate the bead ratio with an experiment similar to the one in this helpful blog post (the gel). I tested 50 bp DNA ladder with a range of volume ratios from 0.5X to 1.5X and ran the product on the Bioanalyzer. This is what I got:



    So basically, the total yield drops off at about 0.7X and lower, but there's no size specificity at any ratio.

    What is going on?

    Details of the experiment:
    • Each starting sample: 500 ng 50 bp ladder (NEB #N3236S), in 10 µL TE (aliquotted from master mix)
    • Add 5 to 15 µL Agencourt RNAClean XP bead mix (I believe this is identical to Ampure XP, except it's RNase-free), depending on ratio, and mix by pipetting
    • Incubate 5 minutes at room temperature
    • Separate beads on magnet 2 minutes and remove supernatant
    • Wash twice in 200 µL fresh 70% ethanol
    • Air-dry 15 minutes
    • Resuspend in 10 µL TE
    • Separate beads 2 minutes and run 1 µL on Bioanalyzer, along with 1 µL of the untreated master mix

  • #2
    Maybe the concentration of PEG in the RNA Ampure beads is higher than in the DNA Ampure beads?

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    • #3
      Originally posted by pmiguel View Post
      Maybe the concentration of PEG in the RNA Ampure beads is higher than in the DNA Ampure beads?
      Unlikely. But in fact, this is the third time I've run this experiment. The first two times, I cut the mix with variable amounts of homemade bead buffer (20% w/v PEG 8000, 2.5 M NaCl, 10 mM Tris-HCl pH 8.0) in order to hold constant the amount of beads and the total reaction volume, and just adjusted the ratio of buffer to TE. I consistently got this result, so I thought I must have misunderstood the chemistry and repeated it this way, which is more standard. So, it's not the buffer. I'm pretty sure they are exactly the same beads, just more expensive because they're certified RNase-free.

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      • #4
        Between 5 ul and 15 ul, you are looking at a very small difference of total PEG between each ratio. PEG % is very important.

        Increase the beads you are using 10x might give you a better result.

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        • #5
          Actually there should be a difference between 0.5X (5µL, 6.67% PEG) and 1.5X (15µL, 12% PEG).
          Unless your beads are out of date or been incorrectly stored, I would suggest there must be a difference, other than just RNase free, between the RNA and DNA beads.

          Thinking logically, why would you want to size select RNA? If you are purifying RNA/cRNA/cDNA for any expression quantification assay you'd want to minimise bias (i.e. betweeen small and large transcripts). My guess is it's probably been formulated not to have size selection.

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          • #6
            Originally posted by TonyBrooks View Post
            Thinking logically, why would you want to size select RNA? If you are purifying RNA/cRNA/cDNA for any expression quantification assay you'd want to minimise bias (i.e. betweeen small and large transcripts). My guess is it's probably been formulated not to have size selection.
            I don't want to size-select RNA at the moment; I just happen to have a lot of these beads because they're required for other purposes and I assumed I could use the same ones for everything. But maybe it might be useful after RNA fragmentation (heat-shearing with magnesium).

            I'm not sure either brand of Agencourt beads is really formulated with size selection in mind. If you look at their website it says nothing about that. I'll try asking their tech support, but I'm pessimistic since they don't even advertise it for size selection.

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            • #7
              My point is, I don't see a need for size selection to be "built in" to the RNA beads, so it's possible they're formulated not to.
              In fact, as you point out, if you use them for purifying magnesium sheared RNA then you specifically DON'T want them to size select.
              DNA beads on the other hand need some degree of size selection in order to reduce smaller dsDNA species (i.e. primer-dimer).

              Comment


              • #8
                What I ended up doing a lot in an effort to reuse beads after one reaction and before another was to take the original beads, put then in the magnet, and discard the buffer, and then start fresh with my own solution of PEG/NaCl. You can try this to simply confirm the beads themselves are not the issue and that whatever is causing it to not work is in the original buffer itself.

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                • #9
                  I just got off the phone with Beckman-Coulter tech support, who assured me that the RNAClean XP bead mix is exactly the same composition as AMPure XP, except prepared under RNase-free conditions. Indeed they don't make any guarantees about size selection with either kit, since they weren't developed with size selection in mind - that would be the SPRIselect kit instead. They're going to take a look at my data, but I remain pessimistic.

                  I may just try this again with a new batch of bead mix altogether.

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                  • #10
                    We size select our libraries all the time with AMPure XP beads. Theoretically the volume should not matter , but try the size selection with the 500ng in 50 to 100 ul, pipet the beads carefully , mix well , and we wait 15 min at room temp , wash , and elute 5 min. with 25ul EB. Usual yield with 0.5 vol. is 700 bp + and 0.7 vol. yields 300bp+.

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                    • #11
                      Update: It was actually the ladder. Apparently there's something weird about the binding chemistry of the NEB ladder, and Beckman Coulter has had this problem before. This protocol also has a cryptic note to that effect. So I tried again with an Invitrogen 100 bp ladder, and although the Bioanalyzer was a bit messy, I can definitely see a size specificity now.

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                      • #12
                        Good know In case we need to titrate a new bottle of beads

                        Thank You

                        Comment


                        • #13
                          Some ladders are made by digesting a plasmid with restriction endonuclease. This leaves single stranded overhangs (often 4 base overhangs). So some ladders have double and single stranded character. Other ladders are completely double stranded (blunt ended). This might be the reason for the discrepancy between ladders.

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                          • #14
                            If restriction fragmented DNA can not be sized using Ampure beads, then sheared DNA can also not be sized using these beads.
                            Since this is a standard procedure I don't think a 4 nt single stranded overhang disturbs DNA or RNA sizing using beads.

                            Comment


                            • #15
                              Originally posted by HeinKey View Post
                              If restriction fragmented DNA can not be sized using Ampure beads, then sheared DNA can also not be sized using these beads.
                              Since this is a standard procedure I don't think a 4 nt single stranded overhang disturbs DNA or RNA sizing using beads.
                              Sheared DNA is end-repaired before size selection (at least in the TruSeq protocols).
                              Jon

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