For RNA extraction, the intestinal pieces were homogenized in lysis buffer using an Ultra-Turrax T8 homonigenisator and RNA was extracted using the E.Z.N.A total RNA kit (Omega Bio-Tek). The amount and quality of the extracted RNA was evaluated using a lab-on-a-chip Bioanalyzer (Agilent Technologies, CA, USA).
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 a time-course study of small intestinal gene expression from the pre-weaning to the post weaning stage.
Data processing
Log(2) transformed expression measures were calculated with the RMA procedure using the software from www.Bioconductor.org
