title: MCW Rattus norvegicus 2K Array Time Course 2002
status: Public on Nov 17 2005
contact.city: Milwaukee
contact.first: Mingyu
contact.last: Liang
contact.email: mliang@mcw.edu
contact.state: WI
contact.address: 8701 Watertown Plank Rd.
contact.department: Physiology
contact.country: USA
contact.institute: Medical College of Wisconsin
contact.phone: 414-456-8539
organism: Rattus norvegicus
manufacture_protocol: The custom microarrays were manufactured by the Department of Physiology at the Medical College of Wisconsin.
  
  Total RNA was isolated from the renal medulla using the TRIzol reagent (Invitrogen/Life Technologies, Carlsbad, CA). Total RNA of 50 µg was reverse-transcribed to cDNA in a reaction primed by 2 µg of oligo-(dT)12–18. The reverse transcription reaction contained 1x RT buffer (Invitrogen/Life Technologies), 2.5 mM MgCl2, 10 mM dithiothreitol, 500 µM each of dATP, dGTP, and dCTP, 40 µM dTTP, 40 µM of either Cy3-dUTP or Cy5-dUTP (Amersham Pharmacia, Piscataway, NJ), and 7.5 U/µl SuperScript II RT enzyme (Invitrogen/Life Technologies) in a total volume of 20 µl. The reaction was performed at 39°C for 2 h with an additional 75 U of SuperScript II RT enzyme added at the middle of the reaction. After RNase H digestion, the cDNA products that had been labeled with either the Cy3 or Cy5 fluorescent dye were purified using Centri-Sep spin columns (Princeton Separations, Adelphia, NJ) and QIAquick purification columns (Qiagen, Valencia, CA). Labeled cDNAs were then concentrated using Microcon YM-30 filters (Millipore, Bedford, MA) and hybridized to a microarray at 65°C for 16 h in a hybridization solution containing mouse Cot-1 DNA, 1 µg/µl poly-dA, 1.14 µg/µl yeast tRNA, 3.4x SSC, and 0.3% SDS. Slides were washed and scanned using the ScanArray 5000 [Packard Bioscience (formerly GSI Lumonix), Meriden, CT] to quantify the fluorescent intensity of Cy3 and Cy5 in each spot. Renal medullary gene expression profiles were examined in four comparisons: SSBN13 rats on the low-salt diet compared with SS/Mcw rats on the low-salt diet; SS/Mcw rats on the high-salt diet compared with SS/Mcw rats on the low-salt diet; SSBN13 rats on the high-salt diet compared with SSBN13 rats on the low-salt diet; and SSBN13 rats on the high-salt diet compared with SS/Mcw rats on the high-salt diet. For any two samples (rats) involved in a comparison, one was labeled with Cy3 and the other was labeled with Cy5. The two samples were pooled after labeling and hybridized to a microarray. To control for dye variations, these two samples were labeled again with Cy3 and Cy5 switched between the two samples and hybridized to a second microarray. Thus 4 groups of rats with 3 rats in each group were studied, and 2 hybridizations were performed for each pair of rats, resulting in a total of 24 microarray hybridizations.
  Custom-made cDNA microarrays containing cDNA probes that represented  80% of all currently known rat genes were used in the present study. The majority of these genes have some known biological functions in rats.  cDNA probes for 1,751 genes were printed on this array.
  The experiment was done at 0 and 2-week time points.  For detailes, please refer to the following papers: Physiol Genomics. 2003 Feb 6;12(3):229-37 and Physiol Genomics. 2002 Feb 28;8(2):139-49