Ratref 12 expression beadchip

The microarray analysis (cohort 1) was performed as previously published [28 (link)]. In short, 500 ng of total RNA was converted to double stranded cDNA and this was used to generate biotinylated cRNA probes using the Illumina TotalPrep RNA Amplification Kit. Biotin-labelled cRNA were then hybridized to Illumina RatRef-12 Expression BeadChip (San Diego, CA, USA). Slides were scanned on a BeadStation 500 System using Beadscan software version 3.5.31. No RNA samples were pooled in this analysis, each placental samples was analyzed independently. Samples were hybridized into wells at random. The Array experiment readout was deposited on ArrayExpress and includes gene expression data from microarray and high throughput sequencing studies (ArrayExpress Accession number E-MTAB-1987).
RAS genes from the KEGG Renin Angiotensin System pathway were studied for differential expression using SAM (Microarray Software, Stanford University) analyses, whereby differentially expressed placental genes (i.e. >2 fold expression change) were identified between each of the 4 gestational age groups.

Three independently isolated batches of immature Leydig cells treated with LH and/or MENT in each group were used for the microarray array. The whole genome-expressed RatRef-12 Expression BeadChip from Illumina Inc (San Diego, CA) was used. Each BeadChip contains 21,910 genes, which are mainly selected from the NCBI RefSeq database covering the entire rat transcriptome. As previously reported (24 (link)), probe labeling, hybridization, washing, and scanning were performed according to the manufacturer’s instructions using the Illumina Total Prep kit (Applied Biosystems, Foster City, CA). First strand cDNA was synthesized in a total volume of 20 μl with the supplied reagents. The complete first-strand product was used for second-strand synthesis and then subjected to column purification. The purified product was then used for in vitro transcription using T7 polymerase. Biotin-16-dUTP was incorporated in this step, resulting in a biotinylated complementary RNA (cRNA) probe. An Agilent 2100 bioanalyzer was used to verify the integrity of the probe. The labeled cRNA (750 ng) was hybridized with the array overnight at 58°C in a total volume of 30 μl of hybridization buffer, followed by rigorous washing and scanning after hybridization.

For the microarray analysis, 500 ng of total RNA was converted to double stranded cDNA and this was used to generate biotinylated cRNA probes using the Illumina TotalPrep RNA Amplification Kit. Biotin-labelled cRNA were then hybridized to Illumina RatRef-12 Expression BeadChip (San Diego, CA, USA). Slides were scanned on a BeadStation 500 System using Beadscan software Version 3.5.31. Each of the placental samples was analysed independently. Array experiment readout was deposited on ArrayExpress (ArrayExpress accession number E-MTAB-1987). Overall microarray results were previously published by our group [16 (link)]. For this study, expression of the 7 enzymes Faah, Mgll, Plcd4, Pld1, Nat1, Daglα, and Ptgs2 in the endocannabinoid system was analyzed.

Microarray experiments were carried out at ZAO ''Genoanalytica'', Moscow, Russia. Total RNA was isolated from tissue samples using guanidine thiocyanate [55 (link)]. RNA integrity was assessed by comparison with the rRNA bands obtained in agarose gel electrophoresis under denaturing conditions. RNA was quantified using NanoDrop, and its quality was assessed using an Agilent RNA 6000 Nano Chip. Total RNA (400 ng) was amplified using an Illumina® TotalPrep™ RNA Amplification Kit (Ambion, USA) containing 22,523 probes for a total of 22,228 rat genes selected primarily from the NCBI Reference Sequence database (Illumina, USA). The Illumina RatRef-12 Expression BeadChip was used in accordance with the manufacturer’s instructions. The BeadArray Reader was employed for data acquisition, and the analysis was accomplished with the help of the Genome Studio software (Illumina, USA) using the gene-expression module. The statistical algorithm used in GenomeStudio gene expression analysis is the Illumina Custom error model.

RNA samples from TG versus WT rats and AAV-VEGF-B versus AAV-HSA rats (N = 6 in all groups) were analyzed with the genome-wide Illumina RatRef-12 Expression BeadChip (BD-27-303; Illumina Inc.). Detailed data analyses were performed with the Chipster software as detailed in the supplemental methods (http://chipster.csc.fi) (Kallio et al, 2011 (link)). The gene array data have been deposited in the Gene Expression Omnibus, accession number GSE38457.

Microarray analysis was performed in SHRSP and F344 samples in individual samples, thus no pooled data were analyzed. For gene expression profiling Illumina RatRef-12 Expression BeadChip with 22523 probes per array was used. Biotinylated, amplified RNA for direct hybridization was generated using the Illumina TotalPrep RNA Amplification Kit (Life Technologies) according to the manufacturer’s instructions. Raw data pre-processing was executed with Genome Studio (Illumina, Version 1.9.0) creating the probe and control profile and the sample information file for further analyses in R (R Core Team. R: A Language and Environment for Statistical Computing. 2013) Quality control and ongoing data processing was performed with package 'lumi' [21 (link)]. A log2-transformation and quantile normalization was applied to the data. For differential expression analysis we used the adequately supplied methods from package 'limma' [22 (link)]. T-statistics and log-odds of differential expression with empirical Bayes were computed and adjusted with Benjamini & Hochberg. Microarray data is deposited in the Gene Expression Omnibus and available under accession number GSE53512. For functional annotation Gene Ontology Categories [23 (link)] were used. Pathways are annotated corresponding to the Kyoto Encyclopedia of Genes and Genomes (KEGG) [24 (link)].