Presentation on theme: "Microarray Analysis Allows detection/ comparison of expression levels of multiple genes. What molecule could be used as an indicator of expression?"— Presentation transcript:
Microarray Analysis Allows detection/ comparison of expression levels of multiple genes. What molecule could be used as an indicator of expression?
DNA? No! There are exactly two copies of every* gene in each cell. RNA? Perfect! The number of mRNA transcripts in a cell is proportional to expression level of the corresponding gene. Well, almost perfect...
mRNA is much less stable than DNA Solution: Isolate mRNA from tissue Immediately reverse transcribe Incorporate a fluorescent tag Result= Fluorescently labeled cDNA
5 3 AAAAAA TTTTTTTT Cy5 Fluorescent Tag 53AAAAAA TTTTTTT Isolate RNA from Source A (e.g., yeast cells grown under aerobic conditions) Isolate RNA from Source B (e.g., yeast cells grown under anaerobic conditions) Synthesize cDNAs against the mRNA templates using fluorescently labeled oligo dT primers. Cy3 Fluorescent Tag
Combine the two populations of cDNAs Apply to microarray
Oligonucleotide Synthesis (How arrays are spotted)
Aerobic Respiration Anaerobic Respiration Gene A expressed? yes Gene B expressed? yes Gene C expressed? yesno Gene D expressed? noyes Gene E expressed? yes Compare expression profiles of yeast grown under anaerobic conditions and yeast grown under aerobic conditions. C and D Which gene(s) should be investigated for a potential role in anaerobic respiration?
GeneResult (Color) C4BPA ODC1 SIAT9 FGG HBG1 CYP24 Our Microarray simulates expression levels of the six genes assigned for the Genome Scavenger Hunt Red indicates expression in malignant lung tissue Blue indicates expression in normal lung tissue Color intensity is proportional to expression level.