Affymetrix vs. glass slide based arrays • • • • Affymetrix Short oligonucleotides Many oligos per gene Single sample hybridized to chip • Fixed platform. • Not universally available for each species. • EXPENSIVE! • Glass.
Download ReportTranscript Affymetrix vs. glass slide based arrays • • • • Affymetrix Short oligonucleotides Many oligos per gene Single sample hybridized to chip • Fixed platform. • Not universally available for each species. • EXPENSIVE! • Glass.
Affymetrix vs. glass slide based arrays • • • • Affymetrix Short oligonucleotides Many oligos per gene Single sample hybridized to chip • Fixed platform. • Not universally available for each species. • EXPENSIVE! • Glass slide • Long oligonucleotides or PCR products • A single oligo or PCR product per gene • Two samples hybridized to chip • Flexible platform • LESS EXPENSIVE especially for small microbial genomes Affymetrix Array With 8,000 Genes 16 perfect match oligos 16 mismatch oligos Aharoni and Vorst, in press Spotted Microarrays • DNA representing gene spotted on slide • Direct comparison between two fluorescent labeled RNA samples Competitive hybridization is the key to two-color DNA microarrays! • Competitive hybridization - both samples have the same opportunity to hybridize. PCR vs oligo arrays • PCR (cDNA) • Double stranded – Less specificity • Significant cost and time involved in sample preparation. • PCR products less stable? • Increased signal strength – Intensity doesn’t correlate with expression levels. Products all different sizes. • Long oligonucleotides • Single stranded – More specificity • Synthesized by company at significant cost. • Oligos stable over time. • Less signal strength – Intensity does correlate with expression levels. All oligos similar Tms. Designing oligonucleotides for DNA microarrays • Free software available - OligoArray • Oligo characteristics – – – – Length - governs specificity and sensitivity No secondary structure Fixed GC range Melting temperature • Cost ~ $10 per oligo • Which strand do you use? Printing DNA microarrays • Focus on glass slide based arrays • Direct printing of pre-synthesized oligos on a glass surface. – Use robotics for precision printing. – Many slide chemistries used. • Building of oligonucleotides on a glass surface – Ink-jet methodology, micro mirrors. Labeling RNA or DNA with Cy3 or Cy5. • Cy3 and Cy5 - most often used fluorescent molecules used to label samples for microarray analysis. – Absorb light at one wavelength and emit at another. – Emission and Excitation spectra do not overlap significantly. – In arrays Cy3 and Cy5 are usually false colored green (Cy3) and red (Cy5) for ease of visualization. More labeling • Direct incorporation - incorporates Cy3-or Cy5-dNTP directly into cDNA – RNA to cDNA - reverse transcriptase – DNA to DNA - DNA polymerase – Big problem - Cy3 and Cy5 are not incorporated with same efficiency. • Indirect incorporation - preferred method. – Incorporate an aminoallyl-dUTP molecule during reverse transcription of RNA to cDNA. – Chemically couple Cy3 or Cy5 dye after cDNA is made. – Coupling is efficient with both dyes. Image Analysis • • • • GenePix (Axon) Quantitate genes Normalize data 16 bit image Data from DNA microarray experiments • Expressed as relative expression levels – Cy5/Cy3 – Not absolute expression • Data must be normalized • Data is log2 transformed in most cases • Excel spreadsheets - ~30 columns and 4300 rows of data per experiment. – Data storage and analysis issues. What is a significant change in gene expression? • 2 fold? 5 fold? • Statistical representation of the data – Significance analysis for microarrays – Standard t-tests – Iterative outlier analysis • Biological replicates vs. technical replicates – Biological replicates are essential for generating significant data.