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Heather R. Pelzel, Joel A. Dietz, Kimberly A. Toops, Michael Waclawski, Robert W. Nickells; Development of a Mini-qPCR Array for Mouse Retinal Ganglion Cells using MIQE Guidelines. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3864.
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© ARVO (1962-2015); The Authors (2016-present)
Changes in retinal ganglion cell (RGC) gene expression are important metrics to evaluate the response of these cells to apoptotic and neuroprotective stimuli. We sought to develop a qPCR array to interrogate normal and stress-response gene expression for mouse RGCs that also complied with the Minimal Information required for Quantitative PCR Experiments (MIQE) guidelines.
RGC specific and select stress-response genes were identified from the literature. Primers were designed to bridge introns, contain 60% GC identity, and amplify approximately 200 bp of cDNA. All primers were tested for specificity at a common temperature. All amplimers were cloned and sequenced, confirming identity. Standard curve data was obtained from cloned cDNAs of all target genes. Experimental cDNAs were generated from retinas isolated from eyes after optic nerve crush, including eyes from mice treated with the HDAC inhibitor trichostatin A (TSA) and from different strains that exhibit different susceptibility to optic nerve damage.
Using a 96 well plate, we analyzed (in triplicate) the transcript abundance of 7 ganglion cell specific or enriched genes (Thy1, Brn3b, Sncg, Nrn1, Fem1c, TrkB, Nfl), 4 stress response genes (Hsp27, Gfap, Bim, BclX), 2 axonal regeneration genes (Gap43, Tubb3), and 2 loading control genes (S16, Gapdh), from two independent samples. All gene products exhibited efficient amplification allowing for either relative quantification using the ΔΔCt method after Pflaffl correction or calculation of absolute values from a representative standard curve included on the plate. Test-retest evaluation of the same and different cDNA batches showed less than 1-cycle variation in most transcripts except those with low abundance. Most variation occurred between retinas of different mice. Experimentally, BALB/cByJ mice exhibit greater damage than DBA/2J mice. RGC mRNAs exponentially decay after crush, and TSA injection was able to prevent and even increase expression of RGC specific genes. These outcomes are consistent with literature reports.
Using best practices, as outlined in the MIQE guidelines, this mini-array provides a useful tool to evaluate RGC gene expression. The use of a mini-array provides a straightforward method to assay multiple samples for several genes at once to generate reliable, reproducible data.
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