Paxgene rna tube

About 2.5 ml of blood was collected from each subject in a RNA PAXGene tube at each study time-point (Qiagen, Frederick, MD). Samples were stored at -80°C until further processing. RNA extraction and Affymetrix microarray chips (HG U133 Plus 2.0, Santa Clara, CA) were processed as previously described (13 ). Affymetric GeneChip Command Console (AGCC, 3.0V) was used to scan images during data acquisition.

A total of 2.5 ml of blood was collected from each subject in a RNA PAXGene tube (Qiagen, Frederick, MD). RNA extraction and Affymetrix microarray chips (HG U133 Plus 2.0, Santa Clara, CA) were processed as previously described (Saligan et al., 2013 ). Affymetric GeneChip Command Console (AGCC, 3.0V) was used to scan images during data acquisition. Affymetrix CEL files containing raw intensity data were imported into Partek Genomics Suite 6.6 (Partek Inc., St. Louis, MO), log transformed, and normalized using the robust multiarray average (RMA) algorithm. Because the chips were processed on different days, Partek batch removal analysis of variance (ANOVA) was used to eliminate differences due to batch variation. ANOVA with false discovery rate (FDR) correction was used to identify differentially expressed genes between groups (FDR < 5%). The gene of interest was further confirmed with a TaqMan-based real-time quantitative PCR (RT-qPCR) using gene-specific primers ((Thermo Fisher Scientific, Waltham, MA). Following genomic DNA elimination, first-strand RNA-cDNA PCR template was generated from 150 ng of RNA using the High-capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). RT-qPCR was performed on a QuantStudio 6 Flex instrument (Thermo Fisher Scientific) and gene of interest was normalized to GAPDH endogenous control.

Pain and anxiety from phlebotomy may cause hemodynamic changes; thus, we drew blood as the last step of data collection. Peripheral blood (2.5 ml) was collected to a PAXgene^™ RNA tube containing red blood cell lysis buffer and an RNA stabilizing solution (QIAGEN, Frederick, MD). The collected blood was stored at −80°C until ready for RNA extraction.

Peripheral blood (2.5 ml) was collected in a PAXgene ^™ RNA tube (QIAGEN, Frederick, MD) and stored at −80°C until ready for RNA extraction. RNA was extracted from whole blood using the PAXgene ^™ Blood RNA system (QIAGEN). The quality of the RNA samples was evaluated using an Agilent 4200 Tapestation (Agilent Technologies, US) by the RNA Integrity Number (RIN), and the quantity of the RNA was measured using a Qubit (Life Technologies, US). All of the samples used for this study had excellent purity (A260/A280≥1.9; A260/A230≥2) and showed no visible signs of degradation (RIN ≥ 9). We used the TruSeq Stranded mRNA library prep kit (Illumina, San Diego, US) to generate mRNA-seq libraries. These kits generated high quality libraries for sequencing by fragmentizing the RNA, performing reverse transcription and ligating the indexed adapters. This allowed the individual libraries to be pooled in equimolar fashion, minimizing potential technical bias of run variation. The pooled libraries were then sequenced with an Illumina NextSeq 500 instrument.

Blood samples were collected at the visit during which the routine glucose challenge test was taken, approximately between 24 and 28 weeks gestation. Blood was drawn into a 2.5 mL Qiagen PAXgene RNA tube, containing an RNA stabilizing agent. Collection tubes were maintained at 4 °C and sent to the study laboratory at Dartmouth within 24 h and stored at −80 °C until they were processed for RNA isolation. Samples were thawed and processed in batches using the PAXgene Blood mRNA kit (PreAnalytix 763134), according to the manufacturer’s instructions, and quantified by Nanodrop. In order to remove any contaminating DNA, RNase-free DNase was added during the extraction process. RNA integrity of batch controls was assessed by BioAnalyzer fragment analysis.