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RNA a nd protein expression a nalysis

To understand pathogenesis What genes/proteins are expressed in order to cause disease? To identify poss ible vaccine candidates

RNA and protein expression analysis techniques

Miranda Lo

Overvi ew
RNA techniques Real-time PCR Microarray RNA sequencing Protein techniques 2DGE DIGE (Differential In Gel Electrophoresis) iTRAQ ChIP-chip/ChIP-Seq RNA vs Protein analysis - Things to consider

Transcriptional analysis (transcriptomics) (transcriptomics)

What genes are express ed under specific

conditions?
Specific gene(s) of interest? Gene discovery global analysis

Isolation of leptospiral RNA and post-processing considerations

mRNA highly unstable

Isolation of leptospiral RNA and postprocessing considerations

RNA samples may need to be treated with: MicrobEnrich separate eukaryotic and prokaryotic RNA MicrobExpress remove rRNA MessageAmp amplify mRNA (lots of antisense RNA) PCR to confirm absence of DNA contamination

Important to work on clean surface and change gloves often

RNAlater: Used to treat samples immediately upon harvest to stop transcription and stabilise RNA Not always suitable: Fine for leptospires grown at 30C and 37C but caused 20 C and 39C cultures to lyse resulting in ~10-fold less RNA yield Lyse cells in Trizol immediately; store at -70C (up to 2 y rs) Keep freeze/thaw of purified RNA to minimum

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Re a l time RT -PCR RT Detection of leptospires in clinical and

Real time RT-PCR RT cDNA synthesis using random hexamers: 5 g RNA in 20 l

environmental samples (Sm ythe et al. BMC Infec t Dis, 2002) Analysis of behaviour of specific target genes
When gene target is known To confirm expression data from other methods (microarray, RNA sequencing) SYBR Green PCR mix gene-specific primers Probes eg TaqMan gene-specific probes Need to use internal normaliser genes which do not alter expression under different conditions Eg. flaB, gyrB

reaction cDNA generally diluted 1/50 and 1.5 l used per 20 l real-time reaction Can study many genes Standard curve for each gene (or pair of primers or probe)

Standards fluorescent intensity vs cy cle

Melt curve

Microa rrays
Comparison of two different samples Differences between genomes Comparison of mRNA expression differences PCR products or oligonucleotides representing all

Leptospira microarray - Copenhageni (Cy3) vs (Cy3 La i (Cy5) genomic DNA hybridisation (Cy5
Lai-specific Copenhageni -specific

genes spotted onto glass slides Each s ample labelled with different fluorescent dye
Probes combined and used in competitive

Original annotation rev ised to 3,666 ORFs Oligonucleotides of 70 bases in length designed using ArrayOligoSelector Genes have been spotted in pairs and the grid is duplicated below so that eac h gene is in quadruplicate

hybridisation on s lide
Slide scanned and relative fluorescence measured

Microa rray a nalysis of Leptospira

Low RNA yield from 100 ml culture Average 50 g total RNA from ~5x1010 cells Indirect and direct methods of cDNA labelling to

generate probes require ~20 g of RNA per sample per array

For low amounts of RNA: 3D NA Array 900MPX

kit
0.5-2 g RNA per sample per array

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Leptospiral microarray s tudies

To identify genes potentially involved in virulence Conditions which mimic in vivo signals: Different temperatures (L o et al, Infec t Imm un, 2006; Qin et al,
BMC Mic robiology, 2006)

Pros a nd cons of microarrays

Pros Visualisation of global gene expression patt erns Relativ ely quick to complete simple experiment and obtain dataset Eg. comparison of 2 conditions in triplicate (6 arrays) can be done in one week Cons Cost approx $AUD350 for one hybridisation (slide + coverslips + labelling materials) Low sensitivity Narrow dynamic range Technical variation Availability of slides for strain of interest REDUNDANT! Next generation sequencing now available

Increased osmolarity (Matsunaga et al, Infec t Imm un, 2007) Presence of serum (Patarak ul et al, BMC Microbiology, 2009) Low iron (L o et al, Infec t Imm un, 2010) Characterisation of a regulatory protein (Lo et al,
Infec t Imm un, 2010)

Identification of pos sible vaccine candidates Highly expressed genes encoding potential surfaceexposed proteins that were also conserved between epidemic serovars in China

Ne xt ge neration s equencing
Methods developed to remove the gel separation

Ne xt Ge neration Sequencing
454 LifeSciences sequencing method

portion of sequencing Reduced cost Many reactions carried out simultaneously Up to 200,000 sequencing reactions in a few hours

Credit card sized reaction plates 98% coverage of a bacterial genome sequence in one run

Chemistry is different to Sanger sequencing

DNA synthesis reaction (polymerase, dNTPs etc.) Each base is added sequentially If a base is incorporated, light is produced Light released is proportional to number of bases Camera monitors process, interpreted by computer Process is repeated

Sol exa Technology Hi gh throughput

Faster, cheaper Rapid: Gigabases of sequence per day. Sequencing of whole genomes within days Automated: very high accuracy (99.99%) Sequencing by synthesis different chemistry to Sanger ddNTP chain

termination Laser based reading of sequence

www.micromon.monash.org/dna-s eq-home.html

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Sol exa Sequencing Pros and Cons

Pros:

Fast, cheap, reduced hands-on work Start with 100ng DNA!! No need for cloning, hence no bias for particular clonable regions Practical applications such as whole genome sequencing to identify point mutations Sequencing extinct life forms, or organisms hard to culture in lab Transcriptome analysis sRNA discovery Very little technical variation Cons: Cost per sample

Ne xt Ge neration Sequencing
Nanoporetech Label free s equencing Uses a protein nanopore combined with a

processive enzyme multiplexed on a silicon chip

http://www.youtube.com/watch?v=HbjAMJ ehSlg

Ne xt Ge neration Sequencing
Current leptospiral study Investigation of in vivo expressed genes Kidneys harvested from infected rats RNA sequencing: in vitro vs in vivo RNA abundance Possibility of small RNA discovery Approx $AUD3000 to sequence RNA from 3 samples in duplicate

Protein expression analysis (proteomics)

What proteins are express ed under specific

conditions?
Studies have identified leptospiral proteins that are expressed more highly eg. in vivo Vaccine candidates? Comparison of different strains

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2-D Ge l Electrophoresis ( 2DGE) (2

Separation of proteins by isoelectric point then

2-D Ge l Electrophoresis ( 2DGE) (2

Can cut out individual proteins for identification

separation by mass
3 pH 10

by mass spectroscopy
Better visualisation of differences between

different protein samples

MW

Comparison of in vitro vs host-grown leptospires by

Identification of

immunblot

OMPs (TX-114 prep)

(Cullen et al., Infec t Imm un, 2002)

Identification of in

vivo express ed proteins

(Nally et al., Infec t Imm un, 2007)

(Nally et al., Infec t Imm un, 2007)

2D -DIGE (Differential In Gel Electrophoresis)

Comparison of up to 3 different

i TRAQ peptide quantification basics

Characterisation of a complex mixture of

samples
Samples labelled with fluorescent dyes Loaded onto same gel which removes gel-to-gel variation Individual proteins separated by 2DGE Gel imaging proteins from different samples visualised based on spectral properties of the dyes Individual proteins/spots analysed as peaks
http://dige-proteomics.neuro.duke. edu/2dgel.html

proteins
Protein mixture digested with trypsin Peptide mixture fractionated by 2D liquid chromatography 30 fractions for TX-114 OM preps Fractions subjected to tandem mass spectrometry Proteins identified by MASCOT and Protein Pilot Relativ e quantification enabled by iTRAQ labelling

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iTRAQ - isobaric tags which allow relative

Leptospiral iTRAQ s tudies

Low iron and 37C upshift (Esghi et al, J Proteome Res,
20 09 )

quantitation due to unique reporter ion

Peptides derived from different samples will have same mass During MS/MS reporter ion is released Ratio of peak intensities corresponds to the relativ e abundance ratio of the peptide

Temperature: 30C vs 37C (Lo et al, PLoS Negl Trop Dis,

20 09 )

New iTRAQ tags available allowing comparison

of up to 8 different samples

Li mi tations of proteomics
Stability of proteins during sample processing Loss of proteins due to proteases Proteins may not be amenable to identification

Trans cription vs translation

Low correlation between protein and mRNA

abundance
Temperature studies 25% of differentially expressed proteins also differentially expressed at the mRNA level

by MS
Protein levels may be below detection limits

(L o et al., PLoS Negl Trop Dis, 2009)

Trans cription vs translation

Variability due to mRNA and proteins harvested

mRNA vs protein expression lack of correlation?

Change in mRNA expression does not necessarily correspond

to change in protein abundance

Lack of correlation may be due to factors affecting

from different cultures and at different times?

Studies where mRNA and proteins derived from same samples also show low correlation Study on Desulfovibrio vulgaris showed mRNA abundance alone can explain only 20-28% of the total variation in protein abundance (Nie et al, Biochem Biophys
Res Commun 2006)

mRNA stability protein translation rates stability/turnover of proteins presence of proteases Studies so far have focused on one environmental factor Gene expression may require more than one signal
Activ ities of small non-coding RNAs (sRNAs)

Most of unknown function but several have been found to modulate post-transcriptional expression of OMPs

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Small non-coding RNAs regulation of OMP nonexpression

sRNA 50-250 nt in

ChIP-chip characterise regulatory proteins ChIP Chromatin ImmunoPrecipitation combined with

length
Regulate gene/protein

microarrays (gene chip)

Method for studying all protein-DNA interactions in vivo on a genomic scale Can be used to define complete transcriptional networks Sigma factors in E. coli Identification of DNA sequence motifs for binding

expression
Several characterised in

E. coli which regulate expression of OMPs eg. OmpC Transcription of some sRNA E-dependent
Fine-tuni ng o f OM com position

RNA-seq for sRNA

discovery

(Vogel and Papenfort, Curr Opin Microbiol, 2006)

ChI P-chi p overview

Fi nal points to consider

Interested in express ion changes at both mRNA

and protein levels to understand regulatory pathways

Formaldehyde

ChIP-seq
Sequence

What proteins are expressed in response to stimuli? Role in virulence? Vaccine candidate? Choice of strain, number of culture pass ages and

growth conditions
Sonicate to fragment DNA to ~300-400 bp (Wade et al. (2009), Mol Micro, 65, 21-26)

Drawing conclusions from global express ion data Genes expressed at different stages of infection or in different host tissues? Helps narrow down genes to focus on

Understanding leptospiral pathogenesis

There are still many questions to answer

Thank you