Microarrays, RNAseq And Functional Genomics CPSC265 Matt Hudson

Microarray Technology Relatively young technology – Already mostly obsolete, though. Usually used like a Northern blot – can determine the amount of mRNA for a particular gene Except – a Northern blot measures one gene at a time A microarray can measure every gene in the genome, simultaneously

Recent! History First microarrays developed by Ron Davis and Pat Brown at Stanford Practical microarrays become available for yeast, humans and plants

Why analyze so many genes? Just because we sequenced a genome doesn’t mean we know anything about the genes. Thousands of genes remain without an assigned function. To find genes involved in a particular process, we can look for mRNAs “up-regulated” during that process. For example, we can look at genes up-regulated in human cells in response to cancer-causing mutations, or look at genes in a crop plant responding to drought. Patterns/clusters of expression are more predictive than looking at one or two prognostic markers – can figure out new pathways

Two Main Types of Microarray Oligonucleotide, photolithographic arrays “Gene Chips” Miniaturized, high density arrays of oligos (Affymetrix Inc., Nimblegen, Inc.) Printed cDNA or Oligonucleotide Arrays  Robotically spotted cDNAs or Oligonucleotides Printed on Nylon, Plastic or Glass surface Can be made in any lab with a robot Several robots in ERML Can also buy printed arrays commercially

The original idea A microarray of thousands of genes on a glass slide Each “spot” is one gene, like a probe in a Northern blot. This time, the probes are fixed, and the target genes move about.

Glass slide microarray summary

The process Building the chip: MASSIVE PCR PCR PURIFICATION and PREPARATION PREPARING SLIDESPRINTING RNA preparation: CELL CULTURE AND HARVEST RNA ISOLATION cDNA PRODUCTION Hybing the chip: POST PROCESSING ARRAY HYBRIDIZATION PROBE LABELING DATA ANALYSIS

Robotically printed arrays 1 nanolitre spots um diameter steel spotting pin

Physical Spotting

Reverse Transcriptase Labelling RNA for Glass slides mRNA (control) cDNA Cy3 labelled Reverse transcription mRNA (treated) cDNA Cy5 labelled Cy3 label Cy5 label

Hybridization Binding of cDNA target samples to cDNA probes on the slide cover slip Hybridize for 5-12 hours

Northern blot vs. Microarray In Northern blotting, the whole mRNA of the organism is on the membrane. The labelled “probe” lights up a band – one gene In a microarray, the whole genome is printed on a slide, one “probe” spot per gene. Mixed, labelled cDNA, made from mRNA from the organism, is added. Each probe lights up green or red according to whether it is more or less abundant between the control and the treated mRNA.

LABEL 3XSSC HYB CHAMBER ARRAY SLIDE LIFTERSLIP SLIDE LABEL Humidity Temperature Formamide (Lowers the Tm) Hybridization chamber

Expression profiling with DNA microarrays cDNA “A” Cy5 labeled cDNA “B” Cy3 labeled Hybridization Scanning Laser 1 Laser 2 + AnalysisImage Capture

Image analysis GenePix

Spotted cDNA microarrays Advantages Lower price and flexibility Can be printed in well equipped lab Simultaneous comparison of two related biological samples (tumor versus normal, treated versus untreated cells) Disadvantages Needs sequence verification Measures the relative level of expression between 2 samples

Affymetrix Microarrays One chip per sample Made by photolithography ~500, base probes …unlike Glass Slide Microarrays Made by a spotting robot ~30, base probes Involves two dyes/one chip Control and experiment on same chip

Affymetrix GeneChip Miniaturized, high density arrays of oligos 1.28-cm by 1.28-cm (409,000 oligos) Manufacturing Process Solid-phase chemical synthesis and Photolithographic fabrication techniques employed in semiconductor industry

Selection of Expression Probes Set of oligos to be synthesized is defined, based on its ability to hybridize to the target genes of interest Probes Sequence Perfect Match Mismatch Chip 5’5’ 3’3’ Computer algorithms are used to design photolithographic masks for use in manufacturing

Photolithographic Synthesis Manufacturing Process Probe arrays are manufactured by light-directed chemical synthesis process which enables the synthesis of hundreds of thousands of discrete compounds in precise locations Lamp MaskChip

Affymetrix Wafer and Chip Format 1.28cm 50… 11µm µm Millions of identical oligonucleotides per feature chips/wafer up to ~ 400,000 “features” / chip

Reverse Transcriptase in vitro transcription Labelling RNA for Affymetrix mRNA cDNA Reverse transcription Transcription Biotin labelled nucleotides cRNA

Target Preparation cDNA Wash & Stain Scan Hybridize (16 hours) mRNA AAAA BBBB Biotin-labeled transcripts Fragment (heat, Mg 2+ ) Fragmented cRNA B B B B

GeneChip ® Expression Analysis Hybridization and Staining Array cRNA Target Hybridized Array Streptravidin- phycoerythrin conjugate

Example: Comparing a mutant cell line with a wild type line.

Instrumentation Affymetrix GeneChip System G Scanner 450 Fluidic Station

Microarray data analysis This is now a very important branch of statistics It is unusual to do thousands of experiments at once. Statistical methods didn’t exist to analyse microarrays. Now they are being rapidly developed.

Normal vs. Normal Normal vs. Tumor

Lung Tumor: Up-Regulated Lung Tumor: Down-Regulated

Microarray Technology - Applications Gene Discovery- –Assigning function to sequence –Finding genes involved in a particular process –Discovery of disease genes and drug targets Genotyping –SNPs –Genetic mapping (Humans, plants) –Patient stratification (pharmacogenomics) –Adverse drug effects (ADE) Microbial ID

Why it is becoming obsolete In a word, RNAseq RNAseq uses DNA sequencing to do the same thing. Rather than an array, you just sequence millions of mRNA fragments, then figure out what genes they are from

Why RNAseq only just caught on It’s been around for a long time, called things like SAGE and MPSS. But they were expensive and arrays were cheap. Now, sequencing is as cheap as arrays Also, you need a fully sequenced reference genome for the computer analysis.

What RNAseq / arrays can’t do Tell you anything about protein levels Tell you anything about post-translational modification of proteins Tell you anything about the structure of proteins Predict the phenotype of a genetic mutant

Proteomics A high througput approach to learning about all the proteins in a cell As microarrays are to a Northern blot, proteomics is to a Western blot Two main approaches – 2D gels + MS Protein microarrays

Protein separation: 2-dimensional gel electrophoresis 1st dimension Separation by charge (isoelectric focussing) 2nd dimension Separation by molecular weight (SDS-PAGE) kDa pH 3pH 10 pI Susan Liddel

Proteins extracted from cow ovarian follicle granulosa cells separated on a broad range IPG strip (pH3-10) followed by a 12.5% polyacrylamide gel, silver stained Susan Liddel

Mass Spectrometry FT-MS can tell you residues of sequence, but only from a purified protein Robots pick spots from 2-D gel, load into MS Also, 2-D and 3-D LC

Array-based protein interaction detection

Protein microarrays

The future of microarrays: Still looking good, in areas other than research Used by pharmaceutical companies, medical diagnostics, etc. In the future, just like silicon chips, likely to get cheaper, faster and more powerful It may not be long before they are routinely used to diagnose disease

The future of proteomics: Many people will tell you proteomics IS the future of biology If they can get it to work as well as microarrays, they will be right The problem is, every protein has different chemistry, while all mRNAs are closely comparable At the moment, proteomics is a hot field, but few major biological discoveries have been made with proteomics – many have been made with microarrays