Methods for the Study of Gene
Expression
Gabriela Salinas-Riester
November 2012 Transcriptome Analysis Labor
Microarray and Deep Sequencing Core FacilityUMG
www.microarrays.med.uni-goettingen.de
Methods for the Study of Gene Expression
Microarray_Introduction
Outline
Linking genome-wide analysis to genomic medicine_2011 2/22Gabriela Salinas-Riester
Microarray_Introduction
Methods for the Study of Gene Expression
➢ From genotype to phenotype: the transcriptome
➢ Types of RNAs and their functions
➢ Methods for Study gene expression
➢ Single transcripts
➢ Multiple transcripts simultaneously: NanoString, Microarrays, NGS
➢ What are microarrays and how do they work?
➢ Agilent platform
➢ Experimental Workflow
➢ Sample Quality control
➢ Agilent Microarray Workflow
➢ Data Analysis
Microarray_Introduction
Linking genome-wide analysis to genomic medicine_2011 2/22Gabriela Salinas-Riester Methods for the Study of Gene Expression
Microarray_Introduction
Linking genome-wide analysis to genomic medicine_2011 2/22Gabriela Salinas-Riester Methods for the Study of Gene Expression
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●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?
Microarray_Introduction
Types of RNAs and their functions
Methods for the Study of Gene Expression
Single Transcripts:
● Northern blot
● RNAse protection assay
● Reverse Transcription (RT)-PCR
● Real-time PCR (qPCR)
● In-situ-hybridization
Multiple Transcripts Simultaneously:
● Dot-Blot analysis
● Differential Display, SAGE
● Subtractive hybridization (1996)
● DNA Microarrays (1999)
● NanoString
● Second generation Sequencing (NGS)
Methods for the Study of Gene Expression
NGS 20081953
Microarrays2000
qRT-PCR
PCR 1986 Northern Blot
RPA
Microarray_Introduction
Methods for the Study of Gene Expression
Methods for the Study of Gene Expression
Microarray_Introduction
Methods for the Study of Gene Expression● Northern blot ● 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
v
Agilent Platform
Microarray_Introduction
Methods for the Study of Gene Expression
Advantages:
● Concurrent detection of thousands of genes
● Result is a global answer
● Experiments are fast and easy to perform
● The technique is cost effective
Disadvantages:
● Expression differences are relativ
● Crosshybridization is possible
● Data has to be validated by other methods (eg. qPCR)
● Weak expressed genes may not be detected
● Changes in mRNA expression pattern may not effect protein abundance
Advantages and Disadvantages of microarray techniques
Microarray_Introduction
Methods for the Study of Gene Expression
Agilent Microarray WorkflowMicroarray_Introduction
Methods for the Study of Gene Expression
Day 1
Day 2
Data Analysis
1.Sample preparation
2.Quality Control
3.Sample labeling
and Amplification
6.Scan/Image (Raw Data)
5.Wash & Stain
Hybridization
v
4.Quality Control
Microarray_Introduction
Experimental Workflow
Methods for the Study of Gene Expression
acRNA
Fragmented acRNA
Total RNATotal RNA
NanoChip (Agilent)
Sample Quality ControlMicroarray_Introduction
Methods for the Study of Gene Expression
Bioinformatics
Microarray_Introduction
➢ The bioinformatic analysis include:
➢ Overview of image processing
➢ Logarithmic transformation
➢ Data Normalization
➢ Data Reduction
➢ Hierarchical Clustering
➢ Principal Component Analysis (PCA)
➢ Statistical Analysis➢ List of candidates
➢ 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
Heat Map Analysis for a specific pathway
Microarray_Introduction
Methods for the Study of Gene Expression
