Preparation of RNA and QC for Expression Profiling John Kenny Centre for Genomic Research, University of Liverpool.

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Presentation transcript:

Preparation of RNA and QC for Expression Profiling John Kenny Centre for Genomic Research, University of Liverpool.

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-RIN e

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, , RNA integrity and the effect on the real-time qRT-PCR performance. Mol. Aspects Med. 2-3, , 2006.

QC of RNA-Quantity Bioanalyzer-RIN values Tapestation-RIN e Quantification....ribogreen/Qubit (Invitrogen)/nanodrop

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): e doi: /journal.pone Cui et al. (2010) A comparison between ribo-minus RNA-sequencing and polyA-selected RNA- sequencing. Genomics 96:

Importance of Clean RNA Total RNA Depleted RNA “Clean” RNA “Dirty” RNA

It Depends...available RNA NuGen Ovation kit SMART/MINT amplified cDNA- important 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 cDNA- important 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 Neil Hall Director Andrew Cossins Director Steve PatersonDirector Christiane Hertz-FowlerManager Lesley Parsons Admin Bioinformatics analysts Roy Chaudhuri Rachael Brenchley Luca Lenzi Computer/Software development Xuan Liu Richard Gregory 454 Team Christian Bourne Illumina Team Suzanne Kay Richard Eccles Arrays/Capture systems/Tech Development Margaret Hughes John Kenny Lisa Olohan Lucille Rainbow Sample Receipt, QC Pia Koldkjaer Statistics Yongxiang Fang