Genechip scanner 3000 7g

150 ng of total RNA was prepared for microarray analysis using the GeneChip™ WT PLUS Reagent Kit (Applied Biosystems, Foster City, CA) according to manufacturer’s protocol. The samples were hybridized (16 h) to Clariom™ S Human Arrays (Applied Biosystems, Foster City, CA) in a GeneChip™ Hybridization Oven 645 (Applied Biosystems™). The arrays were washed and stained using the GeneChip™ Hybridization, Wash and Stain Kit (Applied Biosystems, Foster City, CA) in a GeneChip™ Fluidics Station 450 according to manufacturer’s protocol. The arrays were scanned in a GeneChip™ Scanner 3000 7G (Applied Biosystems, Foster City, CA). Quality control and initial analysis, including scatterplots of differentially expressed genes (DEGs), Venn analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, were performed using Transcriptome Analysis Console (TAC) v 4.0.0.25 (Applied Biosystems, Foster City, CA). Clustered heatmaps were plotted using heatmapper [63 (link)] (www.heatmapper.ca), with the average linkage method and Euclidean distance measurement method. The lists of SE, SE-Drug, and SE-HIV DEGs that were obtained from a TAC analysis were subjected to data mining in a Web-based Gene Set Analysis Toolkit (WebGestalt [64 (link)], www.webgestalt.org), from which biological process and molecular function and cellular component gene ontology (GO) terms were obtained.

Cells were seeded in P100 plates for 24 h, and then exposed to 5 µg/mL of AgNPs for 72 h. After the exposure time, mRNAs were extracted using the PureLink^® Invitrogen commercial kit (Invitrogen, Waltham, MA, USA). The purified RNA was stored at −80 °C for further analysis. Samples were processed with GeneChip^® WT PLUS Reagent Kit (Applied Biosystems, Waltham, MA, USA), hybridized with Clariom^TM D Array, human (Applied Biosystems, Waltham, MA, USA) and scanned with a GeneChip^® Scanner 3000 7G (Applied Biosystems, Waltham, MA, USA). Raw data were processed with an RMA algorithm included in Transcriptome Analysis Console (Applied Biosystems, Waltham, MA, USA) for normalization and gene levels analysis. For each experimental condition (control and cells exposed to AgNPs), three microarray experiments corresponding to three independent biological RNA replicates were processed and analyzed. Fold changes between both experimental conditions were calculated as a quotient between the mean of the gene expression signals. Statistical analysis was performed with e-bayes limma, included in the Transcriptome Analysis Console (Applied Biosystems, Waltham, MA, USA).

A total of twelve RNA samples were processed according to the Applied Biosystems™ recommended protocol. Briefly, 100 ng of total RNA was reverse transcribed to produce cDNA/mRNA hybrid molecule, which was subsequently used as a template to create double-stranded cDNA. This double-stranded cDNA was then amplified via in vitro transcription (IVT) to produce cRNA. In vitro transcription (IVT) generated cRNA was then purified and subjected to 2nd-cycle single-stranded sense cDNA synthesis which was later fragmented, labeled, and hybridized to Human Clariom S Array for 16 hrs at 45 °C with rotation at 60 rpm. Arrays were then washed and stained using the FS450_0007 fluidics protocol and scanned using an Applied Biosystems™ GeneChip™ Scanner 3000 7G.

150 ng total RNA was used to generate amplified and biotinylated sense-strand cDNA with the GeneChip WT PLUS Reagent Kit (Affymetrix), according to the manufacturer’s instructions. cDNA was hybridized to GeneChip Mouse Transcriptome Array 1.0 (Affymetrix, Thermo Fisher Scientific) for 16 hr in a 45°C incubator, rotated at 60 rpm. After hybridization, the microarrays were washed, and then they were stained using the Fluidics Station 450 followed by scanning with the Affymetrix GeneChip Scanner 3000 7G. The data were analyzed in Expression Console software from Affymetrix using default analysis settings. Differentially expressed lncRNAs were identified when the paired t test p value was <0.05 and the fold change was greater than 2. The sample size was three mice per group.⁴³ (link)

mRNA expression in whole testes from P14 CT or Cfp1^Stra8 mice (n = 3/group) was compared using an RNeasy total RNA isolation kit (Qiagen) according to the manufacturer’s instructions. Biotinylated cRNA samples were prepared using 500 ng of total RNA according to the standard Affymetrix protocol (Affymetrix, Santa Clara, CA, USA). After fragmentation, 15 μg of RNA was hybridized at 45 °C for 16 h on a GeneChip Mouse Genome 430 2.0 Arrays. GeneChips were scanned using an Affymetrix GeneChip Scanner 3000 7G. The data were analyzed with RMA using Affymetrix default analysis settings and global scaling as the normalization method. The trimmed mean target intensity of each array was arbitrarily set to 100. The normalized and log-transformed intensity values were then analyzed using GeneSpring GX 12.5 (Agilent Technologies, Santa Clara, CA, USA). Hierarchical clustering data were used to cluster groups that behaved similarly across experiments using GeneSpring GX 12.5 (Agilent Technologies). The clustering algorithm was Euclidean distance and average linkage. The statistical significance of differentially expressed genes (DEGs) was determined when the difference in gene expression between Cfp1^Stra8 and wild type had a p value ≤ 0.05.