I'm reading this fantastic article on estimating body time:Molecular-timetable methods for detection of body time and rhythmdisorders from single-time-point genome-wide expression profilesand one of the things that is not very clear to me is how theresearchers estimated which genes are expressed and which ones arenot:
Total RNA was prepared by using Trizol reagent (GIBCO??BRL).cDNA synthesis and cRNA labeling reactions were performed asdescribed (5). Affymetrix high- density oligonucleotide arrays(Murine Genome Array U74A, Version 1.0, measuring 9,977 independenttranscripts) were hybridized, stained, and washed according to theTechnical Manual (Affymetrix). Affymetrix software was used todeter- mine the average difference (AD) between perfectly matchedprobes and single-base-pair-mismatched probes. The AD of each probewas then scaled globally so that the total AD of each microarraywas equal. The resulting AD values reflect the abundance of a givenmRNA relative to the total RNA popu- lation and were used in allsubsequent analyses
I'm not sure if I'm reading this correctly - did the researcherslook at all RNA available in the cells and calculated the levels ofmessenger RNA produced by expressed genes? if not, how can thelevel of expression of a gene be estimated?
I'm reading this fantastic article on estimating body time:Molecular-timetable methods for detection of body time and rhythmdisorders from single-time-point genome-wide expression profilesand one of the things that is not very clear to me is how theresearchers estimated which genes are expressed and which ones arenot:
Total RNA was prepared by using Trizol reagent (GIBCO??BRL).cDNA synthesis and cRNA labeling reactions were performed asdescribed (5). Affymetrix high- density oligonucleotide arrays(Murine Genome Array U74A, Version 1.0, measuring 9,977 independenttranscripts) were hybridized, stained, and washed according to theTechnical Manual (Affymetrix). Affymetrix software was used todeter- mine the average difference (AD) between perfectly matchedprobes and single-base-pair-mismatched probes. The AD of each probewas then scaled globally so that the total AD of each microarraywas equal. The resulting AD values reflect the abundance of a givenmRNA relative to the total RNA popu- lation and were used in allsubsequent analyses
I'm not sure if I'm reading this correctly - did the researcherslook at all RNA available in the cells and calculated the levels ofmessenger RNA produced by expressed genes? if not, how can thelevel of expression of a gene be estimated?
