Transcript Slide 1
Preparation of RNA and QC for Expression Profiling John Kenny Centre for Genomic Research, University of Liverpool. CGR CGR • Experimental Design, Bioinformatics. • Library production/sequence generation: RNA-Seq, SAGE, Fragment, Mate-pair, Sequence Capture: Sure Select, Nimblegen, HaloPlex ChIP-Seq, Methylation Studies, Metagenomics. Method of RNA Extraction? • Trizol/Company extraction kits • Only exception is miRNA.......... QC of RNA-Quality • Bioanalyzer-RIN values • Tapestation-RINe QC of RNA QC of RNA-the importance of quality Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT- PCR. Biotechnol.Lett. 19, 1601-1613, 20062. RNA integrity and the effect on the real-time qRT-PCR performance. Mol. Aspects Med. 2-3, 126139, 2006. QC of RNA-Quantity • Bioanalyzer-RIN values • Tapestation-RINe • Quantification....ribogreen/Qubit (Invitrogen)/nanodrop • http://www.liv.ac.uk/genomic-research/resource-centre/ It Depends................... Methods of rRNA removal • rRNA depletion (%age recovery-method specific) Ribominus-Life Technologies Ribo-Zero-Epicentre • rRNA removal (30-40% recovery) Terminator-Epicentre • PolyA selection (1-5% recovery) Dynabeads-Invitrogen PolyA purist-Ambion • How many rounds of selection? • Bias in expression profiling? Raz et al. (2011) Protocol Dependence of Sequencing-Based Gene Expression Measurements. PLoS ONE 6(5): e19287. doi:10.1371/journal.pone.0019287 Cui et al. (2010) A comparison between ribo-minus RNA-sequencing and polyA-selected RNAsequencing. Genomics 96:259-265. Importance of Clean RNA “Clean” RNA Total RNA Depleted RNA “Dirty” RNA It Depends...available RNA • NuGen Ovation kit • SMART/MINT amplified cDNAimportant protocol steps controlling rounds of amplification NuGen Ovation SMART/MINT cDNA amplification 454 cDNA sequencing • An example of platform-specific considerations. • Roche cDNA sequencing kit • NuGen Ovation kit • SMART/MINT amplified cDNAimportant protocol steps controlling rounds of amplification removal of polyA tail removal of amplification primer sequences DSN Normalisation RNA-Seq on Short-Read Platforms • STaR kit from Ambion for SOLiD - WT and miRNA • Tru-Seq from Illumina • Script-Seq from Epicentre Ambion STaR kit......SOLiD Sequencing Epicentre Script-Seq....Illumina Sequencing TruSeq Strand-Specific Kit Parkhomchuk et al., NAR 2009 NEB miRNA library....Illumina Sequencing • Do not generate miRNA and total RNA-Seq library from the same library prep It Depends... • The nature of your experiment • The sequencing platform employed • The type of library preparation The Centre for Genomic Research www.liv.ac.uk/genomic-research • • • • • Neil Hall Andrew Cossins Steve Paterson Christiane Hertz-Fowler Lesley Parsons Director Director Director Manager Admin Bioinformatics analysts • Roy Chaudhuri • Rachael Brenchley • Luca Lenzi Illumina Team • Suzanne Kay • Richard Eccles Arrays/Capture systems/Tech Development • Margaret Hughes • John Kenny • Lisa Olohan • Lucille Rainbow Computer/Software development • Xuan Liu • Richard Gregory Sample Receipt, QC 454 Team • Christian Bourne Statistics • Yongxiang Fang • Pia Koldkjaer