cDNA Microarrays Neil Lawrence

Schedule Today: Introduction and Background 18 th AprilIntroduction and Background 25 th AprilcDNA Mircoarrays 2 nd MayNo Lecture 9 th MayAffymetrix GeneChips 16 th MayGuest Lecturer – Dr Pen Rashbass 23 rd MayAnalysis methods

Review - Gene Expression The amount of mRNA produced from a particular gene. Different genes express due to –Cell differentiation –Environment changes

DNA Microarrays New technology for measuring gene expression. Two main types. –cDNA microarrays (Synteni/Stanford). –GeneChip ® (Affymetrix)

Affymetrix vs cDNA Technology differs in: How DNA sequences are laid down –spotting vs photolithography Length of DNA sequences that are laid down –Full sequences vs partial sequences.

cDNA Chips Today we focus on cDNA arrays. Advantages: –Don’t need sequence of gene. –Provide DNA library. –Can make your own chips. Disadvantages –Larger arrays. –Requires image processing.

cDNA Microarrays Lay down a full sequence of the gene –Typically 1000s of base pairs long. An arrayer lays down the spots –Spot of genes DNA placed on a glass slide or nylon membrane.

Different solutions Array Spotting DNA in solution

How is the DNA obtained? DNA Libraries

Biological Sample The total mRNA from two different samples is extracted. These two samples may be from a wild-type and a mutant organism. This mRNA is reverse transcribed to produce cDNA (complementary DNA). Achieved using an enzyme - reverse transcriptase.

Sample Prepartion These cDNA pools should represent all the transcribed genes within each sample. Number of copies of each cDNA should be proportional to the relative of mRNA in each sample Reverse transcription occurs in the presence of fluorescently tagged nucleotides. Each cDNA pool is labelled with different fluorescent dyes (or fluorochromes) usually Cy3 (visualised as green) and Cy5 (visualised as red). Incorporation of these dyes can be different.

Sample Hybridisation Labelling should occur so that the amount of each fluorochrome attached to a given cDNA will be proportional to the relative abundance of the gene in the sample. Labelled cDNA are mixed together and hybridzed onto the slide. Set conditions so that cDNA in the mixed pool binds to its corresponding spot on the array, but will not bond to the slide itself or to other spots.

Scanning the Slide Lasers with different wavelengths are used to excite each dye (Cy3 and Cy5). These lasers emit the appropriate wavelength and use a filter system to stop ‘bleed through’ between the two channels. The amount of dye present should be proportional to the gene expression level of the cell. The overall intensity of signal from each dye should be proportional to the gene expression level within each sample.

Processed Microarray

Slide With Grid Layout often each grid is from a different print-tip

Interpretation of Signals As a result: –Predominantly green signal - gene is more abundant in the Cy3 labelled sample. –Predominantly red signal - gene is more abundant in the Cy5 labelled sample. –Yellow signal - gene is equally abundant in both samples. So fluorescent intensity colour for each spot provides information on abundance and relative expression levels.

Image Processing - ScanAlyze dust spot

Image Processing In ML Group we try and obtain uncertainty estimates. We do this using Bayesian statistics. Bayesian demo

Results Loaded into ScanAlyze

Biological Noise Biological noise – Expression levels depend on –Intrinsic Intracellualar factors (The Stage of the Cell Cycle). –Extrinsic factors (Signals from other cells). Expression level depends on complex interaction of activators and inhibitors. Even in tissue culture dishes (with uniform cells) variation of RNA levels within each cell can occur. The Overall expression level is taken from RNA collected from a pool of cells. It will be a combination of all the transcripts of each cell, our understanding of these processes is incomplete, thus the fluctuations often appear to be random and are thus called biological noise.

Experimental Noise Problems with initial spotting on the arrays e.g. not all spots present or the amount spotted is not the same across all spots. Efficiency of dye incorporation into cDNA. Efficiency of hybridization –Differences in the dye properites (how the dye effects hybridisation with the cDNA strand). –How effectively the cDNA samples bind to the elements (spots). –How effectively the background level is reduced by washing –Whether there are any tidemarks left after washing and drying. Dirt/dust on the slide Differences arising from the scanning process. Image Processing Noise If the image is incorrectly processed, i.e. spot locations are not properly specified, the intensities that are extracted will not truly reflect the expression levels. This variation can be random or systematic.

Normalisation During the array preparations technical variations can occur. ―Dye properties. ―Differences in dye incorporation. ―Differences in scanning. Remove these variations. ―Balance the fluorescent intensities of the dyes. ―Allows comparison of expression levels across experiments (arrays).

Global Normalisation Global Normalisation methods assume the two dyes are related by a constant factor Taking logs

Local Normalisations The dye factor is dependent on: ―Spot intensity (A=RG). ―Location on the array. Local normalisation methods: –Intensity dependent. –Print-tip group.

Intensity dependent Visualise the effect: M-A plot Correction of the intensity dependant variations: Picture from: print-tip effect

Print-tip Group Different experiments may use different printing set-up: –Layout of the tips in the print-head of the arrayer. –Differences on the length or opening of the tips. –Deformation. Print-tip normalisation is simply: (print-tip + A) – dependent Normalisation

Conclusions We’ve reviewed cDNA chips. Key issues: –Hybridization process. –Noise Mechanisms. –Normalisation.