Control subject, biopsy taken from the colon, no macroscopic signs of inflammation.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted with Trizol
Label
Phycoerythrin
Label protocol
First strand cDNA was synthesized from 1 ug total RNA by incubation (42C, 1 hr) in a 20 ul reaction volume containing 2.5 mM T7-(dT)24 primer, 50 mM Tris-HCl pH 8.3, 75 mM KCL, 3 mM MgCl2, 10 mM DTT and 500 mM dNTP, 10 U/ml Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA).Second strand cDNA was synthesized directly by adding 91 ul RNase free water, 30 ul 5 times second strand reaction buffer (Invitrogen), 3 ul 10 mM dNTP, 1 ul Escherichia coli DNA ligase (10 U/ml), 4 ul E. coli DNA polymerase I (10 U/ml), 1 ul E. coli RNase H (2 U/ml) followed by incubation (2 hr, 16C). The ends of the double-stranded cDNA were polished using T4 DNA polymerase (20 U, 5 min, 16C). The cDNA was purified and concentrated by phenol/ chloroform extraction and ethanol precipitation.
Hybridization protocol
The Affymetrix GeneChip® Hybridization, Wash, and Stain kit was used according to the protocol supplied with the kit.
Scan protocol
The Affymetrix system was used and the protocols supplied by Affymetrix followed.
Description
The sample is a part of an analysis of colonic gene expression in patients with ulcerative colitis
Data processing
Log(2) transformed expression measures were calculated with the RMA procedure using the software from www.Bioconductor.org
