Computational protocol: Microarray analysis of relative gene expression stability for selection of internal reference genes in the rhesus macaque brain

Similar protocols

Protocol publication

[…] Tissue from the arcuate nucleus of the medial basal hypothalamus (MBH), hippocampus (HPC) and amygdala (AMD) of rhesus macaques Macaca mulatta were collected for examination in two separates studies. One study was designed to assess effects of three hormone replacement therapy (HRT) conditions on gene expression in the three brain regions collected, and this gene expression was examined using the rhesus GeneChip®. In the HRT study qRT-PCR was conducted on all tissues in addition to microarray analysis. In a second study (cycle study), gene expression in the three brain regions during early follicular (EF) late follicular (LF) and mid luteal (ML) phases of the menstrual cycles of normally menstruating macaques was examined using the human GeneChip®.In the gene microarray analyses for both the HRT and cycle studies, comparisons of the relative expression stabilities of probe sets annotated for independently assessed human housekeeping genes [] were conducted using NormFinder [] algorithms equipped to process data from microarrays. Because the reliability of the NormFinder algorithm is dependent on the expression stability of the sequences examined [], the most widely used normalizers from the list of housekeeping genes http://www.compugen.co.il/supp_info/Housekeeping_genes.html, as well as RPL13A and ALG9 (the latter two considered noteworthy based on our prior results [] and considered here as part of the "popular normalizer" group) were examined in a separate analyses. Again, in efforts to compare probe sets best suited for use in the NormFinder analysis, in the cycle study we designated probe sets as "most representative" according to criteria described later in the current methods section and analyzed these probe sets separately as well.All analyses from both experiments (Figure ) are summarized as follows:1a) HRT study - qRT-PCR: GABAergic component genes, ALG9, GAPDH and RPL13A expression variability compared using geNorm, NormFinder and BestKeeper algorithms.1b) HRT study - gene microarray -general probe set selection: NormFinder relative expression stability assessments of 1444 rhesus GeneChip® probe sets annotated for housekeeping genes, grouped by brain region or hormone treatment.1c) HRT study - gene microarray - "expressed sequences" from the general probe set selection: NormFinder relative expression stability assessments of 824 probe sets showing above average signal intensity and receiving "present" calls in MAS 5.0 analysis.1d) HRT study - gene microarray - probe set selection for popular normalizers: NormFinder relative expression stability assessments of all rhesus GeneChip® probe sets annotated for 15 "popular normalizers", grouped by brain region or hormone treatment.2a) Cycle study - gene microarray - general probe set selection: NormFinder relative expression stability assessments of 1433 human GeneChip® probe sets annotated for housekeeping genes, grouped by brain region or cycle phase.2b) Cycle study - gene microarray - most representative probe set selection: NormFinder relative expression stability assessments of 544 human GeneChip® probe sets selected for the highest annotation quality among the 1433 probe sets annotated for housekeeping genes, grouped by brain region or cycle phase.2c) Cycle study - gene microarray - popular normalizer probe set selection from among the most representative probe sets: NormFinder relative expression stability assessments of most representative human GeneChip® probe sets annotated for 15 "popular normalizers", grouped by brain region or cycle phase. [...] Primers and probes for prospective internal reference genes ALG9, GAPDH and RPL13A as well as GABA subunit receptors GABRA1, GABRA4, GABRG2, GABRD and GABRE and the enzyme GAD1 were designed using Primer Express 2.0 software (Applied Biosystems, Foster City, CA) from sequence sources listed in Table . Rhesus macaque sequences for GAPDH and RPL13A were confirmed in-house using RT-PCR primers against source sequences to amplify rhesus macaque hippocampal cDNA pooled from several individuals. These PCR products were sequenced using an ABI 3730×l DNA sequencer (Applied Biosystems) according to manufacturer's instructions. […]

Pipeline specifications

Software tools NormFinder, geNorm, BestKeeper, Primer Express
Applications Gene expression microarray analysis, qPCR
Organisms Macaca mulatta, Homo sapiens