Transcription profiling of mouse microdissected kidney structures - U74 platform

ABSTRACT: Our laboratory's interest is in understanding the molecular principles that underlie the regional organization of the mammalian metanephric kidney. Our goal is to generate a detailed spatial map of the cellular expression of selected regulatory genes during mammalian kidney development. The goal of this study is to identify genes expressed during the morphogenesis of the nephron. By profiling two specific developmental nephron structures, we expect to identify genes expressed in both, and unique to each, which suggests they may play a role in the underlying mechanism responsible for the morphogenesis. Experiment Overall Design: Two early nephron structures, the renal vesicle and the s-shaped body, and the collecting duct system, were microdissected from E14.5 kidneys based on morphological criteria. Antibody and lectin staining together with RT-PCR were used for identity verification and for exclusion of contamination by other tissues. Samples are minimally pooled for amplification. A biological replicate is a single minimally pooled sample. RNA isolated from the collected structures was put through two rounds of T7-driven amplification to obtain the microgram quantities of cRNA needed for hybridization to Affymetrix microarrays. Detection of genes previously shown to be expressed in these early structures, such as Wt1, Wnt4 and Pax8, validates the data from the screen. The collecting duct sample serves as a reference sample for comparing the renal vesicle and s-shaped body comparison in this study. We also tested amplification of single s-shaped bodies as compared to pooled samples and found the minimally pooled samples resulted in better data for this study (better sensitivity when inspected for genes known to be expressed in s-shaped bodies).

ORGANISM(S): Mus musculus

SUBMITTER: Mehran Sharghi 

PROVIDER: E-GEOD-6309 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress