strain: 129SvEv gender: Male tissue: whole testes genome/variation: Mll5 -/-
Growth protocol
Testes from freshly euthanized adult male mice (strain 129S6 (129SvEv)) were flash frozen and stored at -80 °C.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from three frozen wild type and age-matched homozygous Mll5-/- testes using QIAzol™ lysis reagent (Qiagen, Maryland, USA). The tissues were homogenized in 800 µL QIAzol and transferred to Phase Lock Gel tubes (2 mL, heavy) (Eppendorf AG, Hamburg, Germany) to which 200 µL chloroform was added. Samples were centrifuged and the aqueous phase was removed and transferred to a new tube with an equal volume of isopropanol. After centrifugation, the supernatant was removed from the precipitated RNA pellets, and the pellets were washed with 70% ethanol. The air-dried RNA was dissolved in RNase/DNase free water and stored frozen at -80 °C. Total RNA samples were analyzed on a NanoDrop (Thermo Fisher Scientific, Waltham, MA) spectrophotometer to determine RNA quality and concentration. One µL of the RNA (was adjusted within a range of 25-500 ng/µL) was analyzed on the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) using the RNA 6000 Nano kit.
Label
biotin
Label protocol
Samples with a resulting total RNA of 1 µg or greater and with a RNA integrity number (RIN) of 7.0 or greater were used and the whole transcript sense target labeling procedure followed the Affymetrix protocol, including the RiboMinus™ rRNA removal step. Three biological replicates were compared for each of the genotypes (wild type and homozygous Mll5-/-) for a total of six experiments.
Hybridization protocol
Labelled RNA was probed on GeneChip Mouse Exon Array 1.0 ST chips (Affymetrix, Santa Clara, California) for gene expression analysis and scanned at the CTAG Facility of the British Columbia Cancer Agency, Vancouver, BC, Canada.
Scan protocol
Array scanning was performed according to the manufacturer's instructions (Affymetrix).
Description
RMA expression value derived from ArrayAssist version 5.0 software; core+extended gene level analysis
Data processing
CEL files were processed using ArrayAssist 5.0. Raw intensity calls were normalized using quantile normalization (Bolstad, 2003) and probeset summarization undertaken with gc-rma (Wu, 2004). In this analysis we considered only transcript level summarization for the core plus extended set of probes on the array (about 800,000 probesets). Outliers with greater than 2.0 fold change between genotypes and level of significance, p < 0.05 were selected for further analysis by quantitative PCR (Q-PCR). ArrayAssist results (Gene and Exon) are available on the series record. Affynetrix Command Console normalized data are presented in the sample tables.
