|Application:||Gene expression microarray analysis|
|Number of samples:||6|
|Release date:||Jun 30 2010|
|Last update date:||Mar 21 2012|
|Dataset link||Mhyo IG Rnd vs MhRnd|
To test for transcripts in IG regions, cDNA was generated from M. hyopneumoniae RNA using random hexamers (IDT; Integrated DNA Technologies, Inc. Coralville, IA) and a set of 129 hexamer oligonucleotide primers designed for M. hyopneumoniae mRNA. A single total RNA sample was split into eight 5 μg samples, and cDNA synthesis was conducted on four of these samples using the random hexamers (IDT); for the other four samples, cDNA was generated using primers designed for M. hyopneumoniae as described by Madsen et al. 2006. In each set of four, two samples were fluorescently labeled with Cy3 and the other two were labeled with Cy5 using an indirect labeling protocol. After labeling and cleanup, all 8 samples were quantified on a Nanodrop ND-1000 spectrophotometer. For each array on the slide, 1 μg of labeled cDNA from each type of primer generation (Cy3 or Cy5, random or specific primer set) was combined and hybridized to a PCR featured array and an oligonucleotide featured array. This resulted in 2 arrays of PCR probes hybridized with dye swaps and 2 arrays of oligonucleotide probes with dye swaps. A second RNA sample was split in four 5 μg samples of total RNA, and the hybridization process was repeated for the oligonucleotide probe array.