Gene expression profiling experiments:

Transcription

1 Gene expression profiling experiments: Problems, pitfalls, and solutions. Heli Borg The Alternatives in Microarray Experiments bacteria - eucaryots non poly(a) + - poly(a) + oligonucleotide Affymetrix in situ total RNA - long cdna, DNA - traditional microarray - ex situ - mrna, ribosomal RNA, trna, snrna 1

2 Basic microarray technique Eucaryots procaryots (bacteria) 2

3 Total RNA - mrna mrna - messenger RNA is a copy of a gene. trna - transfer RNA acts as an adaptor to carry the amino acid elements of a protein to the appropriate place as coded for by the mrna. rrna - ribosomal RNA is one of the structural components of the ribosome. snrna - small nuclear RNA is involved in the machinery that processes RNA's as they travel between the nucleus and the cytoplasm. Hybridization 3

4 Content Primary sources of experimental and biological variation Preparation of bacterial targets for Affymetrix Target preparation with poly(a) + mrna from eucaryotic cells Total RNA target preparation from eucaryotic cells Radioactive labeling for pre-synthesized DNA arrays Data acquisition for DNA array experiments Normalisation options Primary sources of variation (1): Differences among cell samples Control of biological variation isogenic strains with identical background genotype, cell type laser-capture techniques for single cell isolation and amplification Standard conditions of cell culture standard cell-specific media standardized genotype comparison of the same cell line should be agreed 4

5 Primary sources of variation (2): RNA isolation procedures Problems: Variation in individual mrna expression levels Risks during centrifugation: temperature/osmotic stress, nutrient deficiency Solutions: Rapid RNA isolation methods, storage reagents Pooling of RNA from three separate extractions Special issues with bacteria: mrna has no poly(a) + tail and decays rapidly specific primers for each ORF have not been effective Primary sources of variation (3): Technical problems improperly coated slides clogged or damaged printing pins unbound DNA on slide wash solution dried on the slide dehydration of the hybridization solution problems of RNA quality or quantity Microarray troubleshooting: image gallery ooting/troubles_index.htm 5

6 Radioactive labeling for pre-synthesized DNA arrays Radioisotope labeled targets can be measured with greater sensitivity and over greater linear range than fluorophore-labeled targets Technical problems: 50 µm scanning resolution requires at least 250 µm probe sites Radioactive emissions from the neighboring spots Useful method in large-format arrays 1mm diameter probe sites with distance of 2mm Preparation of bacterial targets for Affymetrix Total RNA targets are not an option with in situ synthesized arrays as Affymetrix Contamination with genomic DNA can result in its hybridisation to all of the ORFs and thus obscure the target signals, because stringent hybridization canditions cannot be used Mismatch base pairing between perfect match probes and targets can occur (mismatch probes are used to correct this) Procedure to enrich the mrna to ribosomal RNA ratio gets rid of about 90 % of the ribosomal RNA 6

7 Target preparation with non-poly(a) mrna from bacterial cells (2) Target preparation with poly(a) + mrna from eucaryotic cells Problems with target preparation with poly(a) + mrna from eucaryotic cells efficient oligo(dt) purification requires poly(a) + length at least 20 nucleotides in yeast % of total mrna in poly(a) - form degradation mechanism affected by treatment conditions Total RNA should be used for target preparation 7

8 Total RNA target preparation from eucaryotic cells Minimizes experimental manipulations Common method for total RNA preparation for Affymetrix not applicable missing transcripts with short poly(a) tails The same method used for bacterial cells could be used an extra step is needed to generate antisense crna, because Affymetrix contains sense strand probes Data acquisition for DNA array experiments Software for analysis of the array image 12 packages named in the book more resent list available Background correction, global or local Target intensities correlated with filter address or with gene name possible additional work 8

9 Normalization methods attempt to correct: Number of cells in the sample Total RNA isolation efficiency Efficiency in mrna isolation and labelling Hybridization efficiency Signal measurement sensitivity Normalization options: Normalization to total or ribosomal RNA assumes constant total mrna different cell types or conditions result in varying amounts of total RNA Normalization to housekeeping genes sometimes regulated in cells Normalization to a reference RNA given amount of RNA from another organism absolute level of targets, effective for variances Normalization by global scaling sum of the measurements scaled to a common number assumes constant total mrna 9

10 Some Conclusions Different experiment are usually not comparable (reliable) Sources of variation in DNA array experiments is an intense area of bioinformatics DNA array techniques should become more standardized MIAME (Minimum Information About a Microarray Experiment) The goal is to outline the minimum information required to interpret unambiguously and potentially reproduce and verify an array based gene expression monitoring experiment 10