NEBNext poly(A) mRNA magnetic isolation module

[RemitoAmigo]

Hi guys,

NEBNext poly(A) mRNA magnetic isolation module

Did anyone tried this? impressions? results?

Tx.

Remito

[lnevell]

Hello,
I am a post doc and new to this forum. I recently prepared my first NGS library. I used NEBNext poly(A) mRNA magnetic isolation module with the NEBNEXT ultra directional RNA kit. My Bioanalyzer trace had a peak at >10kb and nothing between 100 bp and 10 kb. I am not sure where I went wrong, if it involved the mRNA selection, heat fragmentation, or Ampure XP cleanup.

[RemitoAmigo]

Have you done a Pico on the bionanalyser after your polyA selection and fragmentation?

Remito

[lnevell]

I am afraid I did not check quality at the end of the polyA selection, just at the end of the entire protocol. I will try that next. But I am confused: If the poly A selection failed there should still be a lot of small fragments (which I do not have) and I do not have 18s or 28s peaks.
Thanks,
Lisa

[RemitoAmigo]

Good question, if the poly A failed...I think you would see anything...because you would of most likely thrown everything out. A bit of adapters maybe!?

[lnevell]

PCR cycles

Hello,
I tested the total RNA and found a RIN = 8.60 and that we were starting with 200 ng of total RNA. Then I tested the mRNA isolated with the poly A bead module and found that there was mRNA present but less than 25 ng (the lower quantitative limit of the kit available in my lab). Then I proceeded through the rest of the protocol and ran the dna on a high sensitivity bioanalyzer chip. Initially I used 15 PCR cycles (the upper limit of the recommended number of cycles in the NEB protocol). There was a .34 pg/ul peak at 82 bp and nothing else. I decided to test whether additional PCR cycles would help. I used the library as the starting sample, added additional PCR master mix and enzyme, ran an additional 12 cycles of PCR, and used the Ampure XP beads to clean up the product. Attached are the before and after bioanalyzer results. I now have an 8.36 ng/ul library with an average insert size of 539 bp. I have very little high molecular weight products. I think this will work.
2100 expert_High Sensitivity DNA Assay_DE13806093_2014-09-19_19-59-17.pdf

2100 expert_High Sensitivity DNA Assay_DE13806093_2014-09-20_12-33-25.pdf
Thank you for your suggestions!
Lisa

[nucacidhunter]

Then I tested the mRNA isolated with the poly A bead module and found that there was mRNA present but less than 25 ng (the lower quantitative limit of the kit available in my lab).

I wonder what method was used for mRNA quantification.

I would not recommend sequencing that library, it most likely is artefact of excessive PCR cycling. A standard Illumina RNAseq library insert size is 150bp (270 bp peak with adapters). A library after 27 PCR cycle even if it is not artefact, will not represent transcripts original expression levels.

[Innovelty]

Hi Inevell,

Don't sequence that library, it isn't real.

I had some struggles with mRNA library preps in which I first got weird double peaks (a product of too many PCR cycles) and then went through a phase where I got no peaks at all, and quantified at less than 0.5 ng/uL on a Qubit fluoremeter.

In the last case, it turned out that my problem was occurring at a bead purification step following cDNA synthesis. I was using AMPure XP beads (which according to Beckman Coulter should have a drying time of less than 30 secs with 80% EtOH) but my protocol called for AMPure beads (a drying time of ~5 minutes). Using a much longer drying time than necessary resulted in "over-dried" beads, which would NOT rehydrate/resuspend correctly in the final buffer. It ended up looking like little flakes of metal swirling around in the solution, never becoming a nice homogenous brown again.

The solution for my problem was to NOT over-dry the beads... and to incubate them in the resuspension buffer for 20 minutes at 40C. The heat helps them resuspend and thus helps the DNA get off the beads.

I notice that the NEBnext protocol has you use AMPure XP beads and dry for TEN MINUTES!! Call Beckman Coulter and ask them about this. That seems pretty crazy to me.