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Microarray_Introduction
Methods for the Study of Gene
Expression
Gabriela Salinas-Riester
November 2012
www.microarrays.med.uni-goettingen.de
Transcriptome Analysis Labor
Microarray and Deep Sequencing Core Facility
UMG
Methods for the Study of Gene Expression
Microarray_Introduction
Outline
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From genotype to phenotype: the transcriptome
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Types of RNAs and their functions
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Methods for Study gene expression
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Single transcripts
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Multiple transcripts simultaneously: NanoString, Microarrays, NGS
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What are microarrays and how do they work?
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Agilent platform
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Experimental Workflow
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Sample Quality control
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Agilent Microarray Workflow
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Data Analysis
Gabriela Salinas-Riester
Methods
for the
Study ofmedicine_2011
Gene Expression 2/22
Linking genome-wide
analysis
to genomic
Microarray_Introduction
Gabriela Salinas-Riester
Linking genome-wide
Methods
analysis
for the
to genomic
Study of medicine_2011
Gene Expression 2/22
Microarray_Introduction
Gabriela Salinas-Riester
Linking genome-wide
Methods
analysis
for the
to genomic
Study of medicine_2011
Gene Expression 2/22
Microarray_Introduction
Types of RNAs and their functions
Long RNAs
Function
mRNA (0.4-10 kb)
Coding genes
lincRNA (>200 bp)
Regulating basal transcription machinery
rRNA (1898-3898 bp)
protein synthesis (28S and 18S)
Small RNAs
Function
snRNA
RNA splicing/Regulation of transcription
snoRNA (60-300 bp)
RNA modification
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C/D box snoRNAs
H/ACA box snoRNAs
miRNA (19-24 bp)
Translational repression, mRNA cleavage
piRNAs (26-31nt) Piwi-interacting RNA
Transposone repression/DNA methylation
tiRNAs (17-18nt) tini RNAs
Associated with TSS in animals
tRNA (73-95 bp)
Translation
rRNA (5.7 S and 5.0 S)
protein synthesis
mt-tRNA
Mitochondrial Translation
siRNA (small interfering RNA)
RNA silencing
gRNA (guide RNA)
RNA editing
TSSa-RNAs (20-90 bp)
Maintenance of transcription?
Methods for the Study of Gene Expression
Microarray_Introduction
Methods for the Study of Gene Expression
PCR
1986
Northern Blot
Microarrays
2000
RPA
qRT-PCR
1953
NGS 2008
Single Transcripts:
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Northern blot
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RNAse protection assay
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Reverse Transcription (RT)-PCR
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Real-time PCR (qPCR)
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In-situ-hybridization
Multiple Transcripts Simultaneously:
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Dot-Blot analysis
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Differential Display, SAGE
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Subtractive hybridization (1996)
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DNA Microarrays (1999)
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NanoString
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Second generation Sequencing (NGS)
Methods for the Study of Gene Expression
Microarray_Introduction
Methods for the Study of Gene Expression
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Northern blot
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Subtractive hybridization
Methods for the Study of Gene Expression
Microarray_Introduction
What are microarrays and how do they work?
Methods for the Study of Gene Expression
Microarray_Introduction
Agilent Platform
v
Methods for the Study of Gene Expression
Microarray_Introduction
Advantages and Disadvantages of microarray techniques
Advantages:
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Concurrent detection of thousands of genes
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Result is a global answer
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Experiments are fast and easy to perform
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The technique is cost effective
Disadvantages:
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Expression differences are relativ
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Crosshybridization is possible
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Data has to be validated by other methods (eg. qPCR)
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Weak expressed genes may not be detected
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Changes in mRNA expression pattern may not effect protein abundance
Methods for the Study of Gene Expression
Microarray_Introduction
Agilent Microarray Workflow
Day 1
Day 2
Data Analysis
Methods for the Study of Gene Expression
Microarray_Introduction
Experimental Workflow
6.Scan/Image (Raw Data)
1.Sample preparation
v
5.Wash & Stain
Hybridization
2.Quality Control
3.Sample labeling
4.Quality Control
and Amplification
Methods for the Study of Gene Expression
Microarray_Introduction
Sample Quality Control
Total RNA
acRNA
NanoChip (Agilent)
Fragmented
acRNA
Methods for the Study of Gene Expression
Microarray_Introduction
Bioinformatics
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The bioinformatic analysis include:
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Overview of image processing
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Logarithmic transformation
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Data Normalization
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Data Reduction
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Hierarchical Clustering
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Principal Component Analysis (PCA)
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Statistical Analysis
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List of candidates
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Data Visualization (Heatmaps)
Methods for the Study of Gene Expression
Microarray_Introduction
Data Normalization
Methods for the Study of Gene Expression
Microarray_Introduction
Hierarchical Clustering
Methods for the Study of Gene Expression
Microarray_Introduction
Principal Component Analysis
Methods for the Study of Gene Expression
Microarray_Introduction
Heat Map Analysis for a specific pathway
Methods for the Study of Gene Expression