title: Rat teratogenicity custom array
status: Public on Jan 16 2014
contact.city: Hannover
contact.first: Juergen
contact.last: Borlak
contact.email: Borlak.Juergen@mh-hannover.de
contact.state: Lower Saxony
contact.address: Feodor-Lynen-Str. 35
contact.department: Centre for Pharmacology and Toxicology
contact.country: Germany
contact.institute: Hannover Medical School
contact.phone: +49 511 532 7249
organism: Rattus norvegicus
manufacture_protocol: The cDNA array was prepared using defined 200- to 400-bp fragments of selected and sequence-verified cDNAs. These probes were generated by RT-PCR (Superscript II; Invitrogen, Groningen, The Netherlands), cloned into the pGEM-T vector (Promega, Mannheim, Germany) and spotted in duplicate on different positions on the array (Thum and Borlak, 2008). In brief, the inserts were amplified using vector-sequence-derived primers with the sense primer carrying a 5′-amino modification. Amplified inserts were purified (Qiaquick 96 PCR BioRobot Kit; Qiagen), checked on an agarose gel and diluted to a concentration of 100 ng/Xl. Two nanolitres of diluted probe was spotted by dispensing 20 drops of 100 pl each as duplicates on treated glass slides with a customised ink jet spotter. Probes were re-hydrated for 2 h in a humidified chamber and blocked. As a qualitative measurement for the validity of the data and to check for the uniformity of the hybridization process, the standard deviation of the two ratios for the respective gene was calculated.