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Is anyone aware of any new kits/methods for miRNA library prep? Our current process is long and complicated involving polyacrylamide gel purifications etc. and is often troublesome because a couple highly expressed miRNAs dominate. Is there a company with a new technology that I can't find?
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The kit from NEB has worked well for us using only Ampure XP size selection. BIOO scientific also has a new kit that uses random sequences for the adaptors which gives less bias in ligations. If your highly expressed miRNA is favored in ligation that might help. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3489589/ -
NEXTflex Small RNA Sequencing Kit v2
Hi mRNA123,
Since you are seeing a few miRNAs dominate your results, it definitely sounds like you could benefit from reduced bias. As Chipper mentioned, the NEXTflex Small RNA Sequencing Kit v2 from Bioo Scientific is the only kit for Illumina platforms to offer reduced ligase bias using adapters with randomized ends. This kit is likely much more streamlined than your current protocol, requiring only one PAGE purification of the final product, which is barcoded, double-stranded DNA. This means it is not necessary to leave empty lanes between samples to avoid cross-contamination and you do not have to cut out and elute an RNA product that you may not even be able to see on the gel.
If you have any questions please feel free to PM me, or even better ask them in this thread so that others can see the questions/answers as well.
For disclosure, I am an employee of Bioo Scientific, and I was heavily involved in development of this kit.Comment
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Does anyone know of any kits for miRNA library prep for small amounts of total RNA (100-900 ng)?Comment
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We were having the same problem with a few miRNAs dominating our libraries made with the TruSeq small RNA or NEBNext small RNA library prep kits. We gave the BioO NEXTflex Small RNA Sequencing Kit v2 kit a try a few weeks ago to see if the random end adapters would help. I think there is less bias in libraries we made with the BioO kit compared to libraries made at the same time with the TruSeq kit. However, a few of the BioO libraries didn't work at all, and all of the TruSeq libraries did. I'm not sure, but I think it might be because of the cleanup step after the 3' ligation in the BioO kit. The libraries with less starting material were the ones that failed using the BioO kit.Comment
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There was a nice poster from TriLink Biotech at ASHG 2014 about very low quantity small mRNA (miRNA) library prep. They've developed and patented chemically-modified oligos to prevent adapter dimer formation. It seems they've just signed an agreement with NEB to make their own kits, so they will likely hit the market in 6 months or so. Some details and a link to their poster are here: http://being-bioreactive.com/2014/11...-library-prep/Comment
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You might be interested in the new CAST method of libraries prop from miRNAsOriginally posted by mRNA123 View Post
It is as simple as a conventional RT-PCT techniqueComment
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Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...03-22-2024, 06:39 AM
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