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, 126-139, 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 TechnologiesRibo-Zero-Epicentre

• rRNA removal (30-40% recovery)Terminator-Epicentre

• PolyA selection (1-5% recovery)Dynabeads-InvitrogenPolyA 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.0019287Cui et al. (2010) A comparison between ribo-minus RNA-sequencing and polyA-selected RNA-sequencing. Genomics 96:259-265.

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 amplificationremoval of polyA tailremoval 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 Researchwww.liv.ac.uk/genomic-research

• Neil Hall Director• Andrew Cossins Director• Steve Paterson Director• 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