Computational protocol: Significant changes in endogenous retinal gene expression assessed 1 year after a single intraocular injection of AAV-CNTF or AAV-BDNF

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Protocol publication

[…] A Custom SABiosciences 96 genes PCR array system (Custom RT Profiler array CAPR 10704R) (Qiagen) was used which included a rat genomic DNA contamination control, reverse transcription control, and a positive PCR control (). The genes were categorized into four groups based on Qiagen functional gene groupings: (i) growth factors, cytokines, and receptors; (ii) cell growth and differentiation; (iii) survival and plasticity, and (iv) cell signaling and transcription factors. The PCR reactions were carried out using RT2 SYBR Green ROX FAST Mastermix (Qiagen) according to the manufacturer’s protocols. Arrays were run on a Corbett Rotor Gene 6000 instrument over the course of 2 weeks, using the recommended settings. RNA was considered DNA-free when 40 cycles of real-time PCR did not give an amplification signal for the genomic DNA. All genes were detected and successfully amplified with the exception of Ocm in sample “CNTF rat 5, nasal left eye”; while Lif and Ntrk1 in sample “GFP rat 3, temporal left eye”. Further outliers were identified using qBase+ (Biogazelle, Ghent, Belgium) and excluded from statistical analysis: “Bdnf in BDNF rat 1, nasal” and “BDNF rat 1 temporal”, and “Socs3 in CNTF rat 2, nasal”.Genorm used to find genes with the highest stability (M), using the qBase+ software package (Biogazelle). For each comparison the highest six stable genes were selected. For comparison and confirmation of selected reference genes BestKeeper version 1 Microsoft Excel spreadsheet was also used. Subsequently data were normalized with the verified reference genes Tfrc, Gsk3b, Jak2, and Rpl13a.The results from the samples and selected housekeepers were used in the Qiagen RT2 Profiler PCR Array Data Analysis Rotor-Gene Q Template v1.0 (September 10, 2010) Microsoft excel spreadsheet. This spreadsheet was used to generate a QC report on each array. Fold change (2−ΔΔCT) were normalized using Tfrc, Gsk3b, Jak2, and Rpl13a (with a Genorm M-value <0.03). The P-values calculated in the Qiagen RT2 Profiler PCR Array Data Analysis Rotor-Gene Q Template v1.0 were based on a Student’s t-test.In further analysis the geometric mean of the expression for all genes was calculated relative to normalized expression data as outlined. Because of the significant involvement of the Jak2 gene in both BDNF and CNTF pathways, the Grb2 (Genorm M-value of 0.034) gene was used as an alternative normalization gene to compare expression changes. In comparison analysis the relative expression was calculated using the ddCt method. All statistical tests were performed using SPSS Statistics 19.0 (IBM, Armonk, NY). Left and right eye comparisons and nasal versus temporal comparisons were analyzed using t-tests. Changes between all three groups were assessed by Kruskal–Wallis (nonparametric) and differences between paired groups were assessed by Mann–Whitney U-tests (nonparametric) (significance levels *P < 0.05, **P < 0.01). Fold-changes of the genes were calculated as mean values of 2−∆∆CT or 1/2−∆∆CT relative to controls and were converted to log2 fold changes for use in graphs.After qBase software normalization using the same housekeeper genes as those used previously for nasal-temporal comparisons, data were analyzed using the nonparametric (ranked based) Kruskal–Wallis H-test for effect of treatment for each gene, followed by the nonparametric Mann–Whitney U-test for pairwise comparisons between treatments for significantly affected (P < 0.05) genes. […]

Pipeline specifications

Software tools qbase+, SPSS
Applications Miscellaneous, qPCR
Organisms Rattus norvegicus
Diseases Neurodegenerative Diseases