Groups of 6 mice were administered, daily for 7 consecutive days, with 50, 200, and 400 mg/kg vancomycin, delivered via the iv and ip routes. Six control mice were administered a similar volume of 0.9% saline solution using both routes.
Growth protocol
BALB/c mice were maintained on Purina Certified Rodent Chow 5002 (Richmond, IN, USA) and purified tap water ad libitum in microisolator cages under controlled lighting (12 h light/dark cycle).
Extracted molecule
total RNA
Extraction protocol
Approximately 15-20 mg of each kidney to analyze was transferred into 1 ml Trizol reagent (Invitrogen, Carlsbad, CA, USA) along with a stainless steel bead (Qiagen, Valencia, CA, USA) and homogenized with a TissueLyser (Qiagen). Total RNA was extracted with 200 µl chloroform, precipitated with 500 µl 100% ethanol, and transferred to a RNeasy mini spin column (Qiagen) for purification according to the manufacturer’s instructions. Total RNA was subsequently precipitated with ethanol, resuspended in water and stored at –70°C.
Label
Biotin
Label protocol
cDNA was prepared and cleaned from 10 µg total RNA using the One-Cycle cDNA Synthesis and Clean-up module kits (Affymetrix, Santa Clara, CA, USA). The cDNA was further processed to biotin-labeled cRNA using the IVT labeling kit (Affymetrix). Labeling protocol was performed according to the manufacturer’s instructions, as described in the Affymetrix Genechip Expression Analysis Technical Manual, revision 5 (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
Hybridization protocol
The cRNA was fragmented before hybridizing to the GeneChip Mouse Genome 430 2.0 Array (Affymetrix). Hybridization was performed according to the manufacturer’s instructions, as described in the Affymetrix Genechip Expression Analysis Technical Manual, revision 5 (http://www.affymetrix.com/support/technical/manual/expression_manual.affx).
Gene expression analysis of the kidney of mouse treated with 7 repeat daily doses of vancomycin at 400 mg/kg administered via the intravenous route (iv)
Data processing
The Affymetrix CEL files were loaded into GeneSpring GX version 7.3.1 (Agilent technologies, Santa Clara, CA, USA) and preprocessed using GenChip Robust Multi-Array analysis (GC-RMA).
