title: 11K_VJF-ARRAY
status: Public on Mar 01 2006
contact.city: Paris
contact.first: Sandrine
contact.last: Imbeaud
contact.email: sandrine.imbeaud@inserm.fr
contact.address: 15 rue de l'Ecole de Médecine
contact.department: INSERM UMRS1138-EQUIPE 28, Sorbonne Université-Inserm-Université de Paris
contact.country: France
contact.institute: Centre de Recherche des Cordeliers
organism: Homo sapiens
description: Quality Control of the probes: Aliquots of each PCR product is analyzed for quality by UV measurement and agarose gel electrophoresis (< 2%) with ethidium bromide staining. Sample amplification is considered to have failed when multiple bands, unexpected product size or low integrity is observed. A low A260:A280 ratio is indicative of a low purity of the PCR product and may be associated with alcohol, detergent and protein contaminations, resulting in a high absorbance at 235 nm and 320 nm. These products were flagged out and specifically re-amplified. A re-sequencing step was randomly performed for 689 clones distributed across the plates, in order to identify any manipulation errors and to allow refinement of the annotations. Representative PCR products were quantified using PicoGreen dyes (Molecular Probes, USA) in a fluorescent assay measuring double-strand DNA concentration according to the manufacturer’s instruction. Samples with quantity under 4 pmol, a threshold for our microarrays, are considered to have failed, were flagged out and then re-amplified.
  
  Microarray printing design: The 11K_VJF-array contains 8388 clones from the Mammalian Gene collection (MGC, IMAGE Consortium). This collection, namely IRAL, seeks to identify and sequence a representative full open reading frame clone for each human gene. A complementary cDNA IMAGE clone collection containing 1126 more clones were selected to represent a cancer targeted-set. Both Amersham Biosciences Lucidea™ Universal Scorecard™ and Lucidea™ Microarray ScoreCard v1.1, control clones are also present on this array.
  
  Thus, the 11K_VJF-array consists in a 23,040 spots array (n = 2), including 9,514 spots of cDNA clones (83 %), from which 9,160 non redundant clones, then 432 technical control fragments (4 %) and 1,574 blank spots (13 %).
  
  Arrays printed on this device contain four meta-columns and twelve meta-rows, thus there are 48 grids in total, each grid containing 22 rows and 22 columns. Duplicate spots are placed in the same relative position in two separate meta-columns i.e. meta-column 2 is a replicate of meta-column 1 and meta-column 4 is a replicate of meta-column 3. Overall array dimensions are 53 mm length and 18 mm width. Spot size of each element is around 170 µm in diameter and spot centers are 185 µm apart.
manufacture_protocol: Probes synthesis: The cDNA clone inserts were PCR amplified twice using vector-specific primers (The PCR primers M13: 5’-GTAAAACGACGGCCAGT-3’ and M13 reverse: 5’-AACAGCTATGACCATG-3’ were used for amplification of cDNA inserts in pOTB7 and pDNR-LIB plasmids. The PCR primers T3: 5’-AATTAACCCTCACTAAAGGG-3’ and T7: 5’-GTAATACGACTCACTATAGGGG-3’ were used for amplification of cDNA inserts in Bluescript® SK- plasmids). PCR products were cleaned up by filtration using robotics stations (Multiscreen™ PCR, Millipore, USA; BioRobot 8000, Qiagen, Germany) and eluted in water.
  Quality checked PCR (size, purity, sequence) were pooled, then dried down. A total of 4 pmol of each product were re-suspended into 384-well plates and were combined 1:1 with DMSO to reach the printing criteria.
  
  Printing: Amino-modified mirrored glass slides (type 7star) were printed using the high speed robotics with the 4th generation Lucidea™ array spotter (Amersham Biosciences, USA). During the spotting process, environmental conditions [Clean environment, controlled humidity (~ 65%) and temperature (~ 22°C)] were strictly controlled. DNA adhesion to the glass was achieved by irradiation in the Hoefer model UVC500 illuminator (Hoefer, USA) with energy output of 10e6 µJ/cm². Quality of the spotting was checked by prescanning the slides. Microarrays were routinely stored in opaque plastic slide boxes at room temperature (no more than 6 months).