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Supplemental Data 1: Materials and Methods
Patients, methods and quantification of gene expression
Procedure concerning the selection of patients, tissue resections and QPCR analysis are described in
(Pillaire et al., submitted). Briefly, 74 patients who came from many parts of France and underwent
surgery for primary colorectal adenocarcinoma resection were selected. Exclusion criteria were
patients treated with adjuvant therapy and with tumor microsatellite instability (MSI) since MSI positive
cancers are known to be associated with mismatch repair deficiency. The study was approved by the
National Institute of Cancer (INCa) following the recommendations of the National Agency of
Agreement and Evaluation for Health (ANAES) and in agreement with the 2004 French Bioethics law.
For RNA extraction, 40 to 80 frozen sections (5 to 10µm thick) from tumor and adjacent normal
tissues were at once homogenized using RNeasy extraction kit and total RNA was extracted
according to the manufacturer’s instruction (QIAGEN SA, Courtaboeuf, France). The quality of total
RNA was assessed with the Agilent 2100® bio-analyzer (Agilent Technologies, Massy, France) and 1
to 2µg of total RNA were reverse transcribed using the High-Capacity cDNA Archive Kit (Applied
Biosystems, Foster City, CA). All studied genes and four control genes (18S, GAPDH, HPRT,
YWHAZ) were amplified in triplicate from tumor and normal samples using the TaqMan Universal
PCR Master Mix and the TaqMan Low Density Array technology (Applied Biosystems). All PCR
amplifications were performed with the TaqMan Low Density Array technology. To normalize gene
expression in the matched tumor and normal samples, the QBase software
(http://medgen.ugent.be/qbase/) was used to test the expression stability of the control genes. For
each sample pair, the two most stable control genes were used by QBase to normalize the
expression of all transcripts. Then the T(tumor) / N(normal) ratios of normalized values were
calculated.
Pillaire MJ, Selves J, Gouraud PA, Danjoux M, Negre V, Do C, Gordien K, Bieth A, Guimbaud R,
Trouche D, Pasero P, Méchali M, Hoffmann JS, Cazaux C A "3R" signature of progression, genetic
instability and negative outcome in colorectal cancer. Submitted for publication.
Statistical Analysis
The major criteria of gene expression analysis in tumors were the individual ratio between
standardized tumor and adjacent normal tissue gene expressions. In order to take into account the
non Gaussian distribution of gene expression, non parametric statistics analyses were used. Binomial
exact tests evaluated the significance of gene over- and under-expression. Correlation between
genes was assessed with a non parametric Spearman’s correlation (Spearman’s rho). All
computations were performed using Stata 9.0 SE at the Clinical Data Management and Statistical
Analysis Platform of the Hospitals and School of Medicine of Toulouse (Tiermips).
Samples preparation and western-blotting
For nuclear extracts preparation, cells were harvested using a hypotonic lysis buffer containing 10mM
Tris (pH8.0), 10mM NaCl, 2mM MgCl2 and protease inhibitors and incubated 5 min on ice. 50µl/ml of
10% NP40 were added to each sample that were incubated 10 min on ice. After centrifugation, the
pellet was re-suspended using a hypertonic buffer containing 20mM Hepes (pH7.9), 420 mM NaCl,
1.5 mM MgCl2, 0.2 mM EDTA and 10% glycerol (all from Sigma-Aldrich). Total cell extracts were
obtained by harvesting the cells in a buffer containing 1% Triton X-100, 2% SDS, 150mM NaCl and
phosphatases/proteases inhibitors in 100mM Tris-HCl (pH 7.4).
After centrifugation of nuclear or total extracts, the supernatants were collected and samples were
quantified using a Dc Protein Assay kit (from Bio-Rad).
For Tip60 analysis, nuclear extracts from each cell lines were prepared and submitted to classical
SDS-PAGE. To analyze p400 protein amounts and PARP cleavage, total cell lysates were prepared
and 10 to 60µg of proteins per lane were separated by NuPAGE® Novex 3-8% Tris-acetate gel (from
Invitrogen SARL). Gels were transferred on a PolyVinylidine DiFluoride (PVDF) membrane. Specific
primary antibodies as well as peroxidase-conjugated secondary antibodies were used according to
standard western blot procedure and peroxidase was then detected by using the (Roche Diagnostics,
Meylan, France).
Supplemental Data 1