All procedures performed on animals were done in accordance with guidelines of the American Physiological Society and were approved by The University of Texas MD Anderson Cancer Center Institutional Animal Care and Use Committee (Richard R. Behringer, IACUC Protocol Number: 02-90-01735). Mice carrying a targeted Lim1 null allele (Lim1lacZ), a Lim1 conditional null allele (Lim1flox) and an Rarb2-Cre transgene in which Cre is expressed in the metanephric mesenchyme of the developing kidney, were used in this study. Lim1lacZ/+ and Lim1flox/flox mice were maintained on a C57BL/6J x 129/SvEv genetic background. Rarb2-Cre transgenic mice were initially generated on a C57BL/6J x SJL/J genetic background. (Reference: BMC Nephrol. 7(1):1, 2006, PMID: 16464245)
Growth protocol
To obtain mouse embryos with metanephric mesenchyme-specific Lim1 deficient (Lim1flox/lacZ; Rarb2-Cretg/+) kidneys, timed matings between Lim1+/lacZ; Rarb2-Cretg/+ males and Lim1flox/flox females were established. Kidney samples of two different embryonic stages (E14.5 and E18.5) were isolated. Their genotypes were assigned unambiguously using real time PCR assays detecting the presence of lacZ and Cre alleles, which were established in the M. D. Anderson Cancer Center DNA Analysis Core Facility. For the E14.5 time point, a total of 71 embryos were collected, 17 of them were genotyped as conditional mutants (Lim1flox/lacZ; Rarb2-Cretg/+). Kidneys from 23 Lim1flox/+; Rarb2Cretg/+ embryos were used as “control” kidneys. For the E18.5 time point, a total of 39 embryos were harvested, 9 of them were genotyped as conditional mutants and 14 of them were genotyped as controls. (Reference: BMC Nephrol. 7(1):1, 2006, PMID: 16464245)
Extracted molecule
total RNA
Extraction protocol
Embryonic kidney tissue from each individual was placed in a separate tube with 0.5 ml TRIzol (Invitrogen, Carlsbad, CA) and stored at –75oC until the corresponding visceral tissue could be genotyped. After a genotype was unambiguously assigned to each individual, TRIzol preserved kidneys of the same genotype were pooled and total RNA was prepared as per the manufacturer’s instructions. Total RNA was then processed using a QIAGEN RNeasy Midi Kit before in vitro transcription-labeling reaction per Affymetrix (Santa Clara, CA) recommendation. Once purified, RNA quality was determined by electrophoretic methods using an agarose gel or analysis using an Agilent Bioanalyzer 2100 (Palo Alto, CA) and by spectroscopy at 260 and 280 nm. (Reference: BMC Nephrol. 7(1):1, 2006, PMID: 16464245)
Label
biotin
Label protocol
Five to forty micrograms of total RNA from each pooled embryonic kidney sample was used to produce the cRNA target for the microarray. The target was created using a reverse transcription reaction to produce cDNA (Supercript Choice System, Gibco), which was subsequently subjected to in vitro transcription with biotinylated cytidine-5’-triphosphate and uridine-5’-triphosphate using the ENZO BioArray High Yield RNA Transcript Labeling Kit to produce biotinylated cRNA. (Reference: BMC Nephrol. 7(1):1, 2006, PMID: 16464245)
Hybridization protocol
The target was then fragmented and hybridized to Mouse Genome 430 2.0 Affymetrix GeneChip Arrays (Affymetrix, Santa Clara, CA) in duplicates using an Affymetrix GeneChip Fluidics Station 400, according to the manufacturer’s standard protocols. (Reference: BMC Nephrol. 7(1):1, 2006, PMID: 16464245)
Scan protocol
The arrays were stained with phycoerythrin-coupled avidin and scanned using a GeneArray Scanner 3000. The resultant output was analyzed using Affymetrix Microarray Suite software and examined for excessive background or evidence of RNA degradation. All microarray processing was performed in the Murine Microarray and Affymetrix Facility at the University of Texas M. D. Anderson Cancer Center. (Reference: BMC Nephrol. 7(1):1, 2006, PMID: 16464245)
Description
After scanning, all probe sets were scaled to a signal intensity of 250 and relative levels of expression of each transcript (signal) were determined using Microarray Suite 5.0 software (Affymetrix). The images of all arrays were inspected for physical anomalies and for the presence of excessive background hybridization. Generally, all array results used in this study were of good quality, and no major manufacturer’s defects or abnormalities were detected. (Reference: BMC Nephrol. 7(1):1, 2006, PMID: 16464245)
