Friday (1/15) computer lab session: Location: 3073 (3rd floor), Department of Computational Biology, BST3, 3501 Fifth Avenue. Time: 9:30-10:45AM Play with R (tutorial) at home before the lab session.
The central dogma of molecular biology: DNA mRNA (messenger) rRNA (ribosomal) tRNA (transfer) Protein Ribosome transcription transcription transcription translation Microarray is a technology to globaly ( simultaneously detecting thousands of genes ) detect mRNA expression level.
Why detect expression level of protein or mRNA?
Cell cycle Cancer cells are malignant cells who don’t die but reproduce rapidly instead. Important to repair problematic mutations during cell division.
Example 1: p53 Pathway (an important tumor suppressor) Cancer cells are malignant cells who don’t die but reproduce rapidly instead. (DNA damaged) http://breast-cancer-research.com/content/pdf/bcr426.pdf
Prediction of a disease: If mechanism known, detecting expression level can help identifying cancer patients (e.g. unusual p53 or Kras expression activity). Exploratory: In general, microarray can help identify candidate genes that contribute to tumor progression and propose hypothesis of the underlying genetic network. Why detect expression level of protein or mRNA?
http://www.escience.ws/b572/L13/north.html Northern Blot (an old technique for measuring mRNA expression) mRNA extracted and purified. mRNA loaded for electrophoresis. Lane 1: size standards. Lane 2: RNA to be tested. The gel is charged and RNA “swim” through gel according to weight. - mRNA are transferred from the gel to a membrane. A labelled probe specific for the RNA fragment is incubated with the blot. So the RNA of interest can be detected. See next page for the details of this step. +
http://www.escience.ws/b572/L13/northupclose.html Norther Blot closeup (color staining) In this simplified cartoon, two mRNAs are bound on the membrane. The complement DNAs of A are prepared with label and are hybridized to all the mRNA on the membrane. The labeled complement DNA will bind to A but not B. After washing and detecting, abundance of the target mRNA can be seen.
See animation of RT-PCR: http://www.bio.davidson.edu/courses/Immunology/Flash/RT_PCR.html RT-PCR (reverse transcription-polymerase chain reaction) http://www.ambion.com/techlib/basics/rtpcr/ real-time RT-PCR
RNA is reverse transcribed to DNA.
PCR procedures can be used amplify DNA at exponential rate.
Gel quantification for the amplified product.
---- an semi-quantitative method. Smaller amount of sample needed.
The PCR amplification can be monitored by fluorescence in “real time”.
The fluorescence values recorded in each cycle represent the amount of amplified product.
---- a quantitative method. The current most advanced and accurate analysis for mRNA abundance. Usually used to validate microarray result.
From: http://www.techfak.uni-bielefeld.de/ags/ai/projects/microarray/ An image example Image analysis is more difficult than Affy array. The probes are spotted by robot instead of synthesized and the exact physical location is not known.
Comparison of cDNA array and GeneChip cDNA GeneChip Probe preparation Probes are cDNA fragments, usually amplified by PCR and spotted by robot. Probes are short oligos synthesized using a photolithographic approach. colors Two-color (measures relative intensity) One-color (measures absolute intensity) Gene representation One probe per gene 11-16 probe pairs per gene Probe length Long, varying lengths (hundreds to 1K bp) 25-mers Density Maximum of ~15000 probes. 38500 genes * 11 probes = 423500 probes
Affymetrix GeneChip One color design cDNA microarray Two color design Why the difference?
Affymetrix GeneChip Photolithography (The amount of oligos on a probe is well controlled) cDNA microarray Robotic spotting (The amount of cDNA spotted on a probe may vary greatly)
Advantage and disadvantage of cDNA array and GeneChip cDNA microarray Affymetrix GeneChip The data can be noisy and with variable quality Specific and sensitive. Result very reproducible. Cross(non-specific) hybridization can often happen. Hybridization more specific. May need a RNA amplification procedure. Can use small amount of RNA. More difficulty in image analysis. Image analysis and intensity extraction is easier. Need to search the database for gene annotation. More widely used. Better quality of gene annotation. Cheap. (both initial cost and per slide cost) Expensive (~$400 per array+labeling and hybridization) Can be custom made for special species. Only several popular species are available Do not need to know the exact DNA sequence. Need the DNA sequence for probe selection.
Fig. End-point attachment orients the DNA while the polymeric coating holds it away from the surface of the slide, making the DNA readily available for hybridization. Codelink’s Gel-matrix
Comparisons cDNA GeneChip Codelink Agilent Probe preparation Probes are cDNA fragments, usually amplified by PCR and spot ted by robot. Probes are short oligos synthesized using a photolithographic approach. 3-D aqueous gel matrix Probes are print ed by Inkjet technology from HP colors Two-color (measures relative intensity) One-color (measures absolute intensity) One-color One- or two-color Gene representation One probe per gene 11-16 probe pairs per gene One probe per gene One probe per gene Probe length Long, varying lengths (hundreds to 1K bp) 25-mers 30-mers 60-mers Density Maximum of ~15000 probes. 38500 genes * 11 probes = 423500 ~57000 ~22000 probes Manufacturer Stanford and many labs. Affymetrix company GE company Agilent company