Supplementary Material s70

SUPPLEMENTARY MATERIAL

FISH analysis

Fluorescence in situ hybridization (FISH) validation experiments were performed on metaphase cells obtained from cultured peripheral blood lymphocytes using routine procedures. BAC clones from the RP11 library were obtained from the BACPAC Resource

Center (BPRC) at Children's Hospital Oakland Research Institute in Oakland (California,

USA) and from the CTD library from Invitrogen. They were labelled using nick-translation according to manufacturer’s recommendations (Abbott, Abbott Park, Illinois, USA) or by random priming using using the Bioprime® Array CGH Genomic Labeling System

(Invitrogen, Carlsbad, USA). Slides were viewed on a Zeiss Axioplan 2 or a Olympus fluorescence microscope and images were captured and analysed using the ISIS analysis system (Metasystems, Altlussheim, Germany).

QMPSF analyses

Primers were designed using Primer Premier software (Premier Biosoft International,

Palo Alto, CA, USA). PCR was performed in a final volume of 25 µL containing 100 ng of genomic DNA, 0.3-0.9 µM of each primer, 200 µM dNTPs, 1.5 mM MgCl2 10 % of DMSO and 1 unit of Taq DNA polymerase (ABgene, Courtaboeuf, France). The PCR consisted of 23 cycles of 94°C for 15 s, 50°C for 20 s, and 72°C for 20 s, preceded by an initial denaturation step of 5 min at 94°C and followed by a final extension of 5 min at 72°C. 28 One µL of the

PCR product was resuspended in a mix containing 1.25 µL of deionised formamide, 0.5 µL of

GeneScan-400 Rox (PE Applied Biosystems, Foster City, CA, USA), and 1 µL of loading buffer. After denaturation for 3 min at 90°C, 2 µL of each sample was loaded on an Applied

Biosystems model 3130 automated sequencer (PE Applied Biosystems, Foster City, CA).

Data were analysed using the Gene Mapper software (PE Applied Biosystems, Foster City,

1 CA). Electropherograms were superimposed to those generated from a normal control DNA by adjusting to the same level the peaks obtained for the HMBS control amplicon. The kinetic of the PCR was controlled by superimposition of the electropherogram of a normal male control DNA and a normal female control DNA, the height of the MECP2 peak being 50% reduced in the male control DNA compared to the female control DNA. The height of the

MEF2C peak of the patients was then compared with that of the normal control with 2 copies of the MEF2C gene.

Multiplex PCR / Liquid Chromatography (MP-LC)

PCR were performed in a 25 μl reaction mixture, containing 10 % DMSO (Sigma-

Aldrich, St Louis, MO, USA), 0.2 mM of each dNTP (Amersham Biosciences,

Buckinghamshire, UK), 2 mM MgCl2, 1.5 U DNA polymerase (AmpliTaq® Gold, Applied

Biosystems, Warrington, UK), 5-10 pmol of each primer and 100 ng genomic DNA, in a

Master Cycler PCR System (Eppendorf, Hamburg, Germany). PCR conditions were composed of an initial step of denaturation at 94°C for 5 min, 24 cycles of denaturation at

94°C for 10 sec, annealing at 50°C for 15 sec and extension at 72°C for 20 sec, and a final step of extension at 72°C for 5 min.29 PCR products were then separated by ion-pair reversed- phase high-performance liquid chromatography on a semi-automated 3500 Wave HS System

(Transgenomic, Omaha, NE, USA) and quantified by fluorescent detection using a post- column intercalation dye, as described by Dehainault et al.30 Data analysis was performed using the NavigatorTM Software (Transgenomic, Omaha, NE, USA), normalisation was achieved with HMBS peak and relative peak intensities for each amplicon directly reflected genomic copy number.

2 Quantitative PCR

Primers were selected within regions of unique sequence and designed with the Primer-

Express V2.0 software (Applied Biosystems, Foster City, CA). Relative quantification was performed on an ABI7900 (Applied Biosystems) in a volume of 25 l containing 10 ng of

DNA and using, as PCR program, 2 minutes at 50°C and 10 minutes at 95°C followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. All samples were run in triplicate. Data were analyzed with the SDS software v1.2.3 of the instrument (Applied Biosystems), exported and further analyzed in Excel using the comparative ΔΔCT method.