Go Back   SEQanswers > Literature Watch

Similar Threads
Thread Thread Starter Forum Replies Last Post
The T4 RNA Ligase in Small RNA Illumina Kit mnkyboy RNA Sequencing 2 05-28-2014 12:57 AM
PubMed: Construction of small RNA cDNA libraries for deep sequencing. Newsbot! Literature Watch 0 01-07-2011 11:30 AM
PubMed: Deep sequencing of the small RNA transcriptome of normal and malignant human Newsbot! Literature Watch 0 01-07-2011 11:30 AM
PubMed: DSAP: deep-sequencing small RNA analysis pipeline. Newsbot! Literature Watch 0 05-19-2010 03:24 AM
PubMed: Analysis of microRNA transcriptome by deep sequencing of small RNA libraries Newsbot! Literature Watch 0 05-13-2010 03:00 AM

Thread Tools
Old 10-18-2011, 03:00 AM   #1
RSS Posting Maniac

Join Date: Feb 2008
Posts: 1,443
Default PubMed: RNA-ligase-dependent biases in miRNA representation in deep-sequenced small R

Syndicated from PubMed RSS Feeds

RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries.

RNA. 2011 Sep;17(9):1697-712

Authors: Hafner M, Renwick N, Brown M, Mihailovi? A, Holoch D, Lin C, Pena JT, Nusbaum JD, Morozov P, Ludwig J, Ojo T, Luo S, Schroth G, Tuschl T

Sequencing of small RNA cDNA libraries is an important tool for the discovery of new RNAs and the analysis of their mutational status as well as expression changes across samples. It requires multiple enzyme-catalyzed steps, including sequential oligonucleotide adapter ligations to the 3' and 5' ends of the small RNAs, reverse transcription (RT), and PCR. We assessed biases in representation of miRNAs relative to their input concentration, using a pool of 770 synthetic miRNAs and 45 calibrator oligoribonucleotides, and tested the influence of Rnl1 and two variants of Rnl2, Rnl2(1-249) and Rnl2(1-249)K227Q, for 3'-adapter ligation. The use of the Rnl2 variants for adapter ligations yielded substantially fewer side products compared with Rnl1; however, the benefits of using Rnl2 remained largely obscured by additional biases in the 5'-adapter ligation step; RT and PCR steps did not have a significant impact on read frequencies. Intramolecular secondary structures of miRNA and/or miRNA/3'-adapter products contributed to these biases, which were highly reproducible under defined experimental conditions. We used the synthetic miRNA cocktail to derive correction factors for approximation of the absolute levels of individual miRNAs in biological samples. Finally, we evaluated the influence of 5'-terminal 5-nt barcode extensions for a set of 20 barcoded 3' adapters and observed similar biases in miRNA read distribution, thereby enabling cost-saving multiplex analysis for large-scale miRNA profiling.

PMID: 21775473 [PubMed - indexed for MEDLINE]

Newsbot! is offline   Reply With Quote

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off

All times are GMT -8. The time now is 03:53 AM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2021, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO