Wt ovation pico rna amplification system

Gene expression analysis was performed with total RNA extracted from seven distinct pools of Lu-pos and Lu-neg cells isolated from mammary glands of BALB/cByJ JAX and C57Bl/6 females. Quality control was performed using the Agilent Bioanalyzer and RNA 6000 Pico total RNA Kit (Agilent). The WT-Ovation™ Pico RNA Amplification System (Nugen) was applied from 1 ng of total RNA to generate sufficient amount of biotinylated cDNA. Samples were hybridized on Affymetrix GeneChip Mouse Genome 2.1ST arrays.
Analyses were made using EASANA® (GenoSplice, www.genosplice.com), which is based on the GenoSplice’s FAST DB® release 2014_2 annotations [33 (link)]. Data were normalized using quantile normalization and a paired Student’s t test was used to compare gene intensities in the different samples. Genes were considered significantly regulated when fold-change between the compared groups was ≥ 1.5 and uncorrected p value ≤ 0.05. The molecular and functional interactions of the genes identified were analyzed with KEGG approach (https://www.genome.jp/kegg/).

Drosophila hearts from 1‐ and 5‐week‐old yw wildtype or yw x GMH5 flies (n = 30) were isolated (Fink et al., 2009), removed and homogenized in TRIzol (Invitrogen Life Sciences). Total RNA was precipitated and purified by miRNeasy mini‐column (Qiagen) according to the manufacturer's instructions. The recovered RNA was subjected to first and second strand cDNA synthesis, amplification, and purification using the WT‐ovation Pico RNA Amplification System (NuGEN), Agencourt RNAClean purification beads (Beckman Coulter), and DNA Clean and Concentrator‐25s columns (Zymo Research). cDNA was fragmented and labeled using the FL‐ovation cDNA Biotin Module V2 (NuGEN).

We completed a comprehensive, microarray analysis of gene expression in liver tissue for 10 patients, each, in the non- and light-alcohol groups. The quality of the isolated ribonucleic acid (RNA) was estimated after electrophoresis using an Agilent 2001 Bioanalyzer (Agilent, Santa Clara, CA). Aliquots (50 ng) of total RNA, isolated from the liver biopsy specimens, were subjected to amplification using the WT-Ovation Pico RNA Amplification System (NuGen, San Carlos, CA), according to the manufacturer's instructions. Approximately 10 μg of complementary deoxyribonucleic acid (cDNA) was amplified from the 50 ng of total RNA, with 5 μg of cDNA used for fragmentation and biotin labeling using the FL-Ovation cDNA Biotin Module V2 (NuGen), according to the manufacturer's instructions. Biotin-labeled cDNA was suspended in 220 μL of a hybridization cocktail (NuGen), with 200 μL of this suspension used for hybridization to the Affymetrix Human 133U Plus 2.0 GeneChip (Affymetrix, Santa Clara, CA) containing 54,675 probes. After stringent washing, the microarray chips were stained with streptavidin-phycoerythrin and probe hybridization was determined using a GeneChip Scanner 3000 (Affymetrix). Data files (CEL) were obtained using the GeneChip Operating Software 1.4 (Affymetrix).

Total RNA from the mammary glands of animals treated with BPA or vehicle was isolated using the RNeasy Lipid Tissue kit (Qiagen Inc., Valencia CA), which uses a combination of Qiazol followed by column extraction. Next, 50 ng of the isolated RNA was used to generate amplified cDNA using the WT-Ovation Pico RNA Amplification System (NuGEN, San Carlos, CA). Five µg of amplified cDNA from each sample were labeled with biotin using the Encore Biotin Module kit (NuGEN) and subsequently hybridized to the Rat Genome 230 2.0 Chip Arrays (Affymetrix). Eight microarray data sets, which represented 4 BPA-treated and 4 control animals, were obtained for each time point (24 microarrays in total). Raw data were generated by the GeneChip Scanner 3000 using the appropriate software. Pairwise analysis between the two treatment groups was performed using the GeneSifter.net on-line service (vizXlabs, Seattle, WA) and gene expression differences with p<0.05 were identified.