Laminin alpha5-deficient mouse embryos at embryonic day 15.5 were removed by caesarean section from day 13.5 to 15.5 of gestation. Segments of intestines were taken and immediately stored in RNA Later (Ambion, Courtaboeuf, France).
Extracted molecule
total RNA
Extraction protocol
1. Total RNA was extracted using the Tri Reagent (Molecular Research Center Inc., Euromedex, France) according to the recommendations of the supplier. 2. Total RNA was subjected to a further purification by an additional phenol/chloroform 5/1 (V/V) extraction. 3. RNA concentration was determined by optical density at 260 nm. The integrity and purity of each RNA sample was checked by microcapillary electrophoresis using the Agilent 2100 Bioanalyser (Agilent Technologies, Massy, France).
Label
Cy3,Cy5
Label protocol
1. The microarray experiments were performed in quadruple with independently prepared RNA from mutant and wild-type embryonic intestines derived from 4 different litters. 2. For each RNA sample, a dye-swap labeling and hybridization was performed using a common reference RNA sample corresponding to 15.5-day embryonic intestines of C57B1/6J mice. 3. Cy3. and Cy5. labeled cDNA probes were prepared by reverse transcription of 10 µg total RNA for 2 h at 42° C [400U SuperScript II from Gibco-BRL (Invitrogen Cell Culture, France), 1X reverse transcription buffer, 0.5mM DTT, 2µg anchored oligodT: T20VVN, 500µM dATP, 500µM dCTP, 500µM dGTP, 100µM dTTP and 0.1mM Cy3. or Cy5-dUTP in a 30µl reaction] 4. The reverse transcription was stopped by a 5 min heating at 70C and RNA was degraded by RNAse A (4µg) for 45 min at 37° C. For quality control, a small fraction of the probes was tested by agarose gel electrophoresis.
Channel 2
Source name
common reference RNA sample corresponding to embryonic intestine
Laminin alpha5-deficient mouse embryos at embryonic day 15.5 were removed by caesarean section from day 13.5 to 15.5 of gestation. Segments of intestines were taken and immediately stored in RNA Later (Ambion, Courtaboeuf, France).
Extracted molecule
total RNA
Extraction protocol
1. Total RNA was extracted using the Tri Reagent (Molecular Research Center Inc., Euromedex, France) according to the recommendations of the supplier. 2. Total RNA was subjected to a further purification by an additional phenol/chloroform 5/1 (V/V) extraction. 3. RNA concentration was determined by optical density at 260 nm. The integrity and purity of each RNA sample was checked by microcapillary electrophoresis using the Agilent 2100 Bioanalyser (Agilent Technologies, Massy, France).
Label
Cy5,Cy3
Label protocol
1. The microarray experiments were performed in quadruple with independently prepared RNA from mutant and wild-type embryonic intestines derived from 4 different litters. 2. For each RNA sample, a dye-swap labeling and hybridization was performed using a common reference RNA sample corresponding to 15.5-day embryonic intestines of C57B1/6J mice. 3. Cy3. and Cy5. labeled cDNA probes were prepared by reverse transcription of 10 µg total RNA for 2 h at 42° C [400U SuperScript II from Gibco-BRL (Invitrogen Cell Culture, France), 1X reverse transcription buffer, 0.5mM DTT, 2µg anchored oligodT: T20VVN, 500µM dATP, 500µM dCTP, 500µM dGTP, 100µM dTTP and 0.1mM Cy3. or Cy5-dUTP in a 30µl reaction] 4. The reverse transcription was stopped by a 5 min heating at 70C and RNA was degraded by RNAse A (4µg) for 45 min at 37° C. For quality control, a small fraction of the probes was tested by agarose gel electrophoresis.
Hybridization protocol
1. The Cy3. and Cy5-labeled reverse transcription reactions were mixed together, and purified and concentrated using the Microcon YM-30 filter (Millipore; Molsheim, France) 2. Cy3. and Cy5-labeled and purified probes were resuspended in 40µl of hybridization solution (50 % formamide, 4X SSC, 0.6 % SDS, 5X Denhardt solution, 0.25 mg/ml of mouse Cot-1 DNA, 1mg/ml salmon sperm DNA and 1mg/ml poly(dA). 3. Before hybridization, this probe solution was incubated for at least 45 min at 46C after a 2 min denaturation at 95C (pre-annealing). 4. The slides were immersed in a prehybridization solution (50 % formamide, 4X SSC, 0.1 % SDS, 0.1 % BSA fraction V) for 30 min at 46C before probe hybridization in a humid chamber overnight at 46C. 5. Prior scanning, three posthybridization washes were performed (first in 2X SSC, 0.2 % SDS for 5 min at 65C; second in 2X SSC for 3 min at room temperature and finally in 0.2X SSC for 3 min at room temperature).
Scan protocol
Arrays were scanned using a ScanArray 4000 (Perkin Elmer, Courtaboeuf, France)
Data processing
1. Raw numerical data were extracted from Cy3 and Cy5 images using Imagene 4.0 (BioDiscovery, El Segundo, CA, USA). Mean values of the fluorescent signal obtained for each spot and each channel were used for data analysis. 2. Intra-slide normalization was performed using a method based on invariant rank genes and LOWESS fitting using the Elea software (Tseng et al. 2001; http://www-microarrays.u-strasbg.fr/Elea/index.html). 3. The normalized log2 ratio (experiment/reference) was calculated for each slide. 4. The resultant log2 ratios were averaged from dye-swap duplicates.
