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RNARNA andand proteinprotein expressionexpression analysisanalysis techniquestechniques Miranda Lo
RNARNA andand proteinprotein expressionexpression
analysisanalysis techniquestechniques
Miranda Lo
OverviewOverview  RNA techniques • Real-time PCR • Microarray • RNA sequencing  Protein techniques
OverviewOverview
 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
Isolation of leptospiral RNA and post-processing considerations  mRNA highly unstable • Important to work
Isolation of leptospiral RNA and post-processing
considerations
 mRNA highly unstable
• 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 30°C
and 37°C but caused 20 °C and 39°C cultures to lyse
resulting in ~10-fold less RNA yield
• Lyse cells in Trizol immediately; store at -70C (up to 2 yrs)
• Keep freeze/thaw of purified RNA to minimum

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RNARNA andand proteinprotein expressionexpression analysisanalysis  To understand pathogenesis • What
RNARNA andand proteinprotein expressionexpression analysisanalysis
 To understand pathogenesis
• What genes/proteins are expressed in order to cause
disease?
 To identify possible vaccine candidates
TTranscriptionalranscriptional analysisanalysis ((transcriptomicstranscriptomics))  What genes are expressed under
TTranscriptionalranscriptional analysisanalysis ((transcriptomicstranscriptomics))
 What genes are expressed under specific
conditions?
• Specific gene(s) of interest?
• Gene discovery – global analysis
Isolation of leptospiral RNA and post- processing considerations • RNA samples may need to be
Isolation of leptospiral RNA and post-
processing 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
ReRe alal timetime RTRT--PCRPCR  Detection of leptospires in clinical and environmental samples (Smythe et
ReRe alal timetime RTRT--PCRPCR
 Detection of leptospires in clinical and
environmental samples (Smythe et al. BMC Infect 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
MicroaMicroa rraysrrays  Comparison of two different samples • Differences between genomes • Comparison of
MicroaMicroa rraysrrays
 Comparison of two different samples
• Differences between genomes
• Comparison of mRNA expression differences
 PCR products or oligonucleotides representing all
genes spotted onto glass slides
 Each sample labelled with different fluorescent
dye
 Probes combined and used in competitive
hybridisation on slide
 Slide scanned and relative fluorescence measured
MicroaMicroa rrayrray analysisanalysis ofof LeptospiraLeptospira  Low RNA yield from 100 ml culture • Average
MicroaMicroa rrayrray analysisanalysis ofof LeptospiraLeptospira
 Low RNA yield from 100 ml culture
• Average 50 μg total RNA from ~5x10 10 cells
 Indirect and direct methods of cDNA labelling to
generate probes require ~20 μg of RNA per
sample per array
 For low amounts of RNA: 3DNA Array 900MPX
kit
• 0.5-2 μg RNA per sample per array

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RealReal timetime RTRT--PCRPCR  cDNA synthesis using random hexamers: 5 μg RNA in 20 μl
RealReal timetime RTRT--PCRPCR
 cDNA synthesis using random hexamers: 5 μg RNA in 20 μl
reaction
• cDNA generally diluted 1/50 and 1.5 μl used per 20 μl
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
LeptospiraLeptospira microarraymicroarray-- CopenhageniCopenhageni (Cy(Cy33)) vsvs LaiLai (Cy(Cy55)) genomicgenomic
LeptospiraLeptospira microarraymicroarray-- CopenhageniCopenhageni (Cy(Cy33)) vsvs
LaiLai (Cy(Cy55)) genomicgenomic DNADNA hybridisationhybridisation
Lai-specific
Copenhageni-specific
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
each gene is in quadruplicate
• Genes have been spotted in pairs and the grid is duplicated below so that each
LeptospiralLeptospiral microarraymicroarray studiesstudies  To identify genes potentially involved in virulence •
LeptospiralLeptospiral microarraymicroarray studiesstudies
 To identify genes potentially involved in virulence
• Conditions which mimic in vivo signals:
 Different temperatures (Lo et al, Infect Immun, 2006; Qin et al,
BMC Microbiology, 2006)
 Increased osmolarity (Matsunaga etal, Infect Immun, 2007)
 Presence of serum (Patarakul et al, BMC Microbiology, 2009)
 Low iron (Lo et al, Infect Immun, 2010)
 Characterisation of a regulatory protein (Loet al,
Infect Immun, 2010)
 Identification of possible vaccine candidates
• Highly expressed genes encoding potential surface-
exposed proteins that were also conserved between
epidemic serovars in China
NextNext gege nerationneration sequencingsequencing  Methods developed to remove the gel separation portion of
NextNext gege nerationneration sequencingsequencing
 Methods developed to remove the gel separation
portion of sequencing
• Reduced cost
• Many reactions carried out simultaneously
• Up to 200,000 sequencing reactions in a few hours
 Chemistry is different to Sanger sequencing • DNA synthesis reaction (polymerase, dNTPs etc.) •
 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

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ProsPros andand conscons ofof microarraysmicroarrays  Pros • Visualisation of global gene expression patterns •
ProsPros andand conscons ofof microarraysmicroarrays
 Pros
• Visualisation of global gene expression patterns
• Relatively 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
NextNext GeGe nerationneration SequencingSequencing  454 LifeSciences sequencing method • Credit card sized
NextNext GeGe nerationneration SequencingSequencing
 454 LifeSciences sequencing method
• Credit card sized reaction plates
• 98% coverage of a bacterial genome sequence in one
run
SolexaSolexa TechnologyTechnology –– HighHigh throughputthroughput  Faster, cheaper  Rapid: Gigabases of
SolexaSolexa TechnologyTechnology –– HighHigh throughputthroughput
 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
NextNext GeGe nerationneration SequencingSequencing  Nanoporetech  Label free sequencing  Uses a protein
NextNext GeGe nerationneration SequencingSequencing  Nanoporetech  Label free sequencing  Uses a protein
NextNext GeGe nerationneration SequencingSequencing
 Nanoporetech
 Label free sequencing
 Uses a protein nanopore combined with a
processive enzyme multiplexed on a silicon chip
NextNext GeGe nerationneration SequencingSequencing  Current leptospiral study • Investigation of in vivo expressed
NextNext GeGe nerationneration SequencingSequencing
 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

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SolexaSolexa SequencingSequencing ProsPros andand ConsCons  Pros: • Fast, cheap, reduced hands-on work • Start
SolexaSolexa SequencingSequencing ProsPros andand ConsCons
 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
 http://www.youtube.com/watch?v=HbjAMJehSlg
 http://www.youtube.com/watch?v=HbjAMJehSlg
ProteinProtein expressionexpression analysisanalysis (proteomics)(proteomics)  What proteins are expressed under
ProteinProtein expressionexpression analysisanalysis (proteomics)(proteomics)
 What proteins are expressed under specific
conditions?
• Studies have identified leptospiral proteins that are
expressed more highly eg. in vivo
 Vaccine candidates?
• Comparison of different strains
22--DD GeGe ll ElectrophoresisElectrophoresis ((22DGE)DGE)  Separation of proteins by isoelectric point then
22--DD GeGe ll ElectrophoresisElectrophoresis ((22DGE)DGE)
 Separation of proteins by isoelectric point then
separation by mass
3
10
pH
MW
 Identification of OMPs (TX-114 prep) (Cullen et al., Infect Immun, 2002)  Identification of
 Identification of
OMPs (TX-114 prep)
(Cullen et al., Infect Immun, 2002)
 Identification of in
vivo expressed
proteins
(Nally et al., Infect Immun, 2007)
22DD--DIGEDIGE ((DDifferentialifferential IInn GGelel EElectrophoresis)lectrophoresis)  Comparison of up to 3
22DD--DIGEDIGE ((DDifferentialifferential IInn GGelel EElectrophoresis)lectrophoresis)
 Comparison of up to 3 different
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

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22--DD GeGe ll ElectrophoresisElectrophoresis ((22DGE)DGE)  Can cut out individual proteins for identification by
22--DD GeGe ll ElectrophoresisElectrophoresis ((22DGE)DGE)
 Can cut out individual proteins for identification
by mass spectroscopy
 Better visualisation of differences between
different protein samples
 Comparison of in vitro vs host-grown leptospires by immunblot (Nally et al., Infect Immun,
 Comparison of in vitro vs host-grown leptospires by
immunblot
(Nally et al., Infect Immun, 2007)
iTRAQiTRAQ peptidepeptide quantificationquantification basicsbasics  Characterisation of a complex mixture of
iTRAQiTRAQ peptidepeptide quantificationquantification basicsbasics
 Characterisation of a complex mixture of
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
• Relative quantification enabled by iTRAQ labelling
 iTRAQ - isobaric tags which allow relative quantitation due to unique reporter ion •
 iTRAQ - isobaric tags which allow relative
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 relative
abundance ratio of the peptide
LimiLimi tationstations ofof proteomicsproteomics  Stability of proteins during sample processing • Loss of
LimiLimi tationstations ofof proteomicsproteomics
 Stability of proteins during sample processing
• Loss of proteins due to proteases
 Proteins may not be amenable to identification
by MS
 Protein levels may be below detection limits
TranscriptionTranscription vsvs translationtranslation  Variability due to mRNA and proteins harvested from different
TranscriptionTranscription vsvs translationtranslation
 Variability due to mRNA and proteins harvested
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
ResCommun 2006)

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LeptospiralLeptospiral iTRAQiTRAQ studiesstudies  Low iron and 37C upshift (Esghi et al, J Proteome Res,
LeptospiralLeptospiral iTRAQiTRAQ studiesstudies
 Low iron and 37C upshift (Esghi et al, J Proteome Res,
20 09 )
 Temperature: 30C vs 37C (Lo et al,
PLoS Negl Trop Dis,
20 09 )
 New iTRAQ tags available allowing
of up to 8 different samples
comparison
TranscriptionTranscription vsvs translationtranslation  Low correlation between abundance protein and mRNA 
TranscriptionTranscription vsvs translationtranslation
 Low correlation between
abundance
protein and mRNA
 Temperature studies
• 25% of differentially expressed proteins also
differentially expressed at the mRNA level
(Lo et al., PLoS Negl TropDis, 2009)
mRNAmRNA vsvs proteinprotein expressionexpression –– lacklack ofof correlation?correlation?  Change in mRNA
mRNAmRNA vsvs proteinprotein expressionexpression –– lacklack ofof correlation?correlation?
 Change in mRNA expression does not necessarily correspond
to change in protein abundance
 Lack of correlation may be due to factors affecting
• 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
 Activities of small non-coding RNAs (sRNAs)
• Most of unknown function but several have been found to
modulate post-transcriptional expression of OMPs
SmallSmall nonnon--codingcoding RNAsRNAs ––regulationregulation ofof OMPOMP expressionexpression  sRNA – 50-250
SmallSmall nonnon--codingcoding RNAsRNAs ––regulationregulation ofof OMPOMP
expressionexpression
 sRNA – 50-250 nt in
length
 Regulate gene/protein
expression
 Several characterised in
E. coli which regulate
expression of OMPs eg.
OmpC
 Transcription of some
sRNA  E -dependent
• Fine-tuning of OM
composition
 RNA-seq for sRNA
discovery
(Vogel and Papenfort, Curr Opin Microbiol, 2006)
ChIP-chip overview Formaldehyde ChIP-seq Sequence Sonicate to fragment DNA to ~300-400bp (Wade et al. (2009),
ChIP-chip overview
Formaldehyde
ChIP-seq
Sequence
Sonicate to fragment
DNA to ~300-400bp
(Wade et al. (2009), Mol Micro, 65, 21-26)
Understanding leptospiral pathogenesis  There are still many questions to answer… Thank you
Understanding leptospiral pathogenesis
 There are still many questions to answer…
Thank you

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ChIPChIP--chipchip ––characterisecharacterise regulatoryregulatory proteinsproteins  Chromatin ImmunoPrecipitation
ChIPChIP--chipchip ––characterisecharacterise regulatoryregulatory proteinsproteins
 Chromatin ImmunoPrecipitation combined with
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
FiFi nalnal pointspoints toto considerconsider  Interested in expression changes at both mRNA and protein
FiFi nalnal pointspoints toto considerconsider
 Interested in expression changes at both mRNA
and protein levels to understand regulatory
pathways
• What proteins are expressed in response to stimuli?
Role in virulence? Vaccine candidate?
 Choice of strain, number of culture passages and
growth conditions
 Drawing conclusions from global expression data
• Genes expressed at different stages of infection or in
different host tissues?
• Helps narrow down genes to focus on