title: harvard_Carpodacusmexicanus_1
status: Public on Feb 24 2012
contact.city: Moulis
contact.first: Camille
contact.last: Bonneaud
contact.email: bonneaud@dr14.cnrs.fr
contact.address: Station d'Ecologie Experimentale
contact.country: France
contact.institute: Centre National de la Recherche Scientifique (CNRS)
organism: Haemorhous mexicanus
manufacture_protocol: Microarray construction
  We constructed a microarray using cDNA clones from the subtraction suppression hybridisation (SSH) libraries constructed in Wang et al. and enriched in cDNA differentially expressed between MG-infected and control house finches 2-weeks post-infection (4).  The microarray consisted of 1000 unique amplicons and included the 220 clones previously identified as significantlydifferentially expressed between control and infected house finches using a macroarray approach (4), as well as 694 randomly selected clones from the librairies, many of whose expression responses to infection are unknown (4).  The clones were collected from the SSH libraries with a toothpick and grown in 1.3 ml of LB + ampicilin at 37°C overnight.  We isolated the plasmid DNA using a Plasmid & BAC extraction kit (AutoGen) on an AutoGenprep 965.  We amplified 5 µl of the eluted DNA in 10 µl Buffer 10 (Lucigen), 0.8 µl dNTP (100 µM), 8 µl of each primer M13/M13R (10 µM), 0.8 µl (4 U) EconoTaq DNA polymerase (Lucigen) and 56.4 µl of sterile dH2O.  The reaction was run for 35 cycles consisting of a 90s 94°C denaturating step, a 45s 50°C annealing step and a 45s 72°C extension step.  PCR products were purified using a QIAGEN MinElute 96 UF Purification kit and run on a 2% agarose gel.  Amplified inserts were subsequently printed onto the array slides.
  We also printed five house finch ‘housekeeping’ genes to help in normalization procedures (Actin related protein 2/3, ATP synthetase, ATPase V1 subunit G1, Basic transcription factor 3, Calmodulin 2; accession numbers: bankit1324533, 1324536, 1324538, 1324542, 1324554).  These genes were generated by amplification of cDNA extracted from house finch spleens (see below), using degenerate primers designed from conserved sequences of humans, mice, chicken and, when available, zebra finches.  We amplified 2 µl of cDNA in 2.5µl Buffer 10 (Lucigen), 2.5 µl dNTP (100 µM), 1 µl of each primer (10 µM), 0.2 µl (2.5U) EconoTaq DNA polymerase (Lucigen) and 15.8 µl of sterile dH2O.  The reaction was run for 35 cycles consisting of a 1 min 94°C denaturating step, a 2 min 60°C annealing step and a 3 min 72°C extension step. PCR products were run on a 2% agarose gel and purified using a QIAGEN MinElute 96 UF Purification kit.  Each gene was amplified twice and while we kept one reaction intact, we ligated and transformed the other into One Shot TOP10 Chemically Competent Escherichia coli using the TOPO Cloning kit (Invitrogen).  Colonies were picked and added to 1.3 ml of LB medium with ampicilin to grow overnight at 37°C.  We then isolated the plasmid DNA using a Plasmid & BAC extraction kit (AutoGen) on an AutoGenprep 965.  We amplified 2 µl of the extracted plasmids in 2.5 µl Buffer 10 (Lucigen), 0.2 µl dNTP (100 µM), 2 µl of each primer M13/M13R (10 µM), 0.2 µl (2.5U) EconoTaq DNA polymerase (Lucigen) and 14.1 µl of sterile dH2O. All PCR products were purified using a QIAGEN MinElute 96 UF Purification kit and run on a 2% agarose gel.  We verified the identity of the purified PCR products by sequencing them on an ABI 377 DNA sequencer (see below).  We printed both the PCR products and the transformed PCR products of the five house finch housekeeping genes onto the array slides.  We quantified purified PCR products on a Nanodrop spectrophotometer.
  Additionally, we printed the PCR products from the DNA amplification of 11 E. coli housekeeping genes (arcA, aroE, dnaE, gapA, gnd, icdA, pgm, polB, putin, trpA, trpB) to serve as external spike-ins(5).  DNA was extracted from a few E. coli colonies obtained above using the QIAGEN DNeasy Tissue and we amplified E. coli house keeping genes as described in (6-7).  PCR products were purified, sequenced and quantified as described above.
  We printed the clones on poly-L-lysine coated glass slides using an OmniGrid 100 (GeneMachines, BST Scientific).  All clones were printed twice on each grid and each grid was replicated twice on each microarray slide.  After printing, the slides were blocked by rehydration, UV crosslinking at 60 mJ, and dipped in blocking solution (6g Succinic Anhydride, 335 ml 1-Methyl-2-pyrrolidinone, 15 ml Sodium Borate).  Denaturing was subsequently performed by dipping the slides in MiliQ water, and then in 95% Ethanol, and spun dry by centrifugation for 2 min at 1000 rpm.  The desiccated slides were stored at room temperature in a closed container until further use.