Total RNA from frozen tissue was isolated by homogenization using Trizol Reagent (Invitrogen, Cergy-Pontoise, France) according to the manufacturer's recommendations. All RNA samples were purified on Qiagen columns according to the manufacturer's protocol (RNeasy Mini kit, Qiagen, Courtaboeuf, France). Quality and integrity of total extracted and purified RNA were determined by Agilent 2100 bio analyser (Massy, France). The RIN of each RNA sample was between 7.5 to 9.5 (84).
Label
Cy3
Label protocol
Twenty microg of each RNA sample was converted into aminoallyl-labelled cDNA with oligo-dT primers, amino-allyl-dUTP (Sigma, Saint-Quentin-Fallavier, France) and superscript II Reverse Transcriptase (Invitrogen, Cergy-Pontoise, France). The cDNA was purified on a Microcon PCR column according to the manufacturer's protocol (Millipore, St-Quentin-en-Yvelines, France) and added to an aliquot of fluorescent dye (Cy3 or Cy5: GE HealthCare, Vélizy, France). Unused reactive sites were blocked by hydroxylamine 4M, and fluorescent probes were purified on QiaQuick columns according to the manufacturer's recommendations (Qiagen, Courtaboeuf, France). Every probe was quantified using Nanodrop spectrophotometer and quality was assessed on an agarose gel electrophoresis 1% TAE 1X. The mean lengths of the fluorescent probes were analyzed by the Typhoon Imaging system scanner (GE HealthCare, Vélizy, France).
Total RNA from frozen tissue was isolated by homogenization using Trizol Reagent (Invitrogen, Cergy-Pontoise, France) according to the manufacturer's recommendations. All RNA samples were purified on Qiagen columns according to the manufacturer's protocol (RNeasy Mini kit, Qiagen, Courtaboeuf, France). Quality and integrity of total extracted and purified RNA were determined by Agilent 2100 bio analyser (Massy, France). The RIN of each RNA sample was between 7.5 to 9.5 (84).
Label
Cy5
Label protocol
Twenty microg of each RNA sample was converted into aminoallyl-labelled cDNA with oligo-dT primers, amino-allyl-dUTP (Sigma, Saint-Quentin-Fallavier, France) and superscript II Reverse Transcriptase (Invitrogen, Cergy-Pontoise, France). The cDNA was purified on a Microcon PCR column according to the manufacturer's protocol (Millipore, St-Quentin-en-Yvelines, France) and added to an aliquot of fluorescent dye (Cy3 or Cy5: GE HealthCare, Vélizy, France). Unused reactive sites were blocked by hydroxylamine 4M, and fluorescent probes were purified on QiaQuick columns according to the manufacturer's recommendations (Qiagen, Courtaboeuf, France). Every probe was quantified using Nanodrop spectrophotometer and quality was assessed on an agarose gel electrophoresis 1% TAE 1X. The mean lengths of the fluorescent probes were analyzed by the Typhoon Imaging system scanner (GE HealthCare, Vélizy, France).
Hybridization protocol
Samples were then combined according to their fluorescent intensity(picomoles), denatured 2 min at 95°C, and co hybridized on the 13 257 oligoarray at 42°C for 16 to 18 hr using the Corning Pronto!TM Universal Microarray Kits according to the manufacturer~s recommendations (Fischer Scientific Bioblock, Illkirsch, France). After stringent washes to remove unbound cDNA, slides were scanned using a GenePix 4000B scanner (Axon instrument, France) and features were analysed with GenePix Pro Version 4.0 software (Axon Instrument).
For the AI versus SCNT comparison, it is made by an indirect comparison for the intercaruncular zones: - with first a comparison between CJ (cycle) versus NT (SCNT) - then CJ (cycle) versus IAJ (AI)
Data processing
Varmixt normalization Data were normalized by a global loess regression
