Microarray Preprocessing

MBP1010H, Department of Medical BiophysicsIrakli (Erik) Dzneladze

Affymetrix microarray processing in R

• Four separate processing steps: background correction, normalization, pm correction and summary expression value computation

• Single Affy function runs selected algorithms in sequence

Affy processing pipeline

Step 1 - background correction

• Scanner image picks up background noise in every image

• Background noise may be due to unbound fluorescent dyes (e.g. Cy3 and Cy5) used to label the RNA on the chip

• This background is quantified and subtracted from probe intensity values

Step 2 - normalization

• The hybridization step cannot be perfectly controlled.

• Event though RNA is quantified prior to hybridization, it is impossible to get the exact same amount of RNA to hybridize to each chip

• The result of this is chip to chip differences in overall distribution of probe intensity values

• The purpose of normalization is to minimize these systematic differences between chips so that individual chips can be compared to each other

Step 3 – pm correction

• Affymetrix GeneChips contain both perfect match (mm) and mismatch (mm) probes

• Mm probes quantify non-specific and cross-hybridization

• Originally mm signal was subtracted from pm signal to correct for non-specific and cross hybridization

• Many researchers prefer to ignore the mm probes entirely and use uncorrected pm probes alone

Step 4 – summary expression value computation

• Each gene is represented by one or more probes sets

• Each probe set includes 11-20 probe pairs

• Expression value for a gene is a summary of corresponding probe-level data

• i.e. probe level intensity values correlated with “gene expression”

Instructions can be found in the manual

Some terms used in the manual

Preprocessing ExampleAgilent Platform (Cy3 and Cy5)