Leukolock total rna isolation system

RNA was isolated from one of the two EDTA tubes using the LeukoLOCK Total RNA Isolation System (Ambion, USA). This system is optimized for use with human blood and offers the isolation of total RNA from the leukocyte population [41 (link)]. In order to maximize RNA isolation yield, all samples were stabilized with RNALater® within 35 minutes of the blood draw. The nucleic acid content was quantified using the Nanodrop spectrophotometer (ThermoScientific). Total RNA quality and quantity was then determined using the Agilent 2100 Bioanalyzer (The Centre for Applied Genomics, Toronto, Canada). The resulting extracted RNA was stored at -80 °C until required for further analysis.

Votavova et al. (2011 (link)) assessed the effect of smoking on maternal cells at the transcription level. In the study, they collected blood samples of 52 nonsmokers and 20 smokers whose ages ranged from 18 to 41. However, only 46 nonsmokers and 19 smokers were engaged for the following analysis. Sample information used in this study is given in Supplementary Table S1. Expression levels of 24,526 transcripts in these samples were detected by the HumanRef-8 v3 Expression BeadChips (Illumina, San Diego, CA, United States). Based on the data (GSE27272), we aimed to screen age-associated mRNAs. Detailed experimental procedures were reported in the study (Votavova et al., 2011 (link)). In brief, RNA samples were extracted and purified from blood samples by using the LeukoLOCK™ Total RNA Isolation System (Ambion, Austin, TX, United States). Second, cRNA was synthesized and biotinylated by using the llumina TotalPrep RNA amplification kit (Ambion). The hybridization reaction of each cRNA sample was conducted on the beadchips and scanned by using the BeadArray Reader. Finally, the obtained raw data were processed and normalized by the quantile method in the Lumi package of R software (www.r-project.org).

Whole blood was collected in EDTA and passed through LeukoLOCK filters (LeukoLOCK Total RNA Isolation System, Ambion Inc. USA). The filters captured the total leukocyte population, while plasma, platelets and red blood cells were eliminated. The filters were flushed with PBS to remove residual red blood cells and then with RNAlater (LeukoLOCK) to stabilize leukocyte RNA. Filters were sealed and stored at -80°C until further processing.

Phenotypic, genealogical, and biological data were collected from individuals belonging to nine populations in Ethiopia and Tanzania. Prior to sample collection, IRB approval for this project was obtained from the University of Pennsylvania. Written informed consent was obtained from all participants and research/ethics approval and permits were obtained from the following institutions prior to sample collection: the University of Addis Ababa and the Federal Democratic Republic of Ethiopia Ministry of Science and Technology National Health Research Ethics Review Committee; COSTECH, NIMR, and Muhimbili University of Health and Allied Sciences in Dar es Salaam, Tanzania. To obtain DNA and RNA data, whole blood was collected using vacutainers and RNA was stabilized in the field using LeukoLOCK Total RNA Isolation System (Ambion life Technologies). The Poly(A)Purist Kit (Ambion Life Technologies, CA) was used for mRNA selection, and Ampure XP magnetic beads (Beckman Coulter, CA) were used for size selection after amplification.

Using the LeukoLOCK™ Fractionation & Stabilization Kit (ThermoFisher Scientific), blood was collected and fractionated from the wing vein of 3-week-old turkeys (from experiment 1) before inoculation as well as 2 dpi with 10¹⁰ CFU S. Heidelberg following NCAH SOP-ARU-0300. RNA from the leukocyte population (white blood cells) was extracted using the LeukoLOCK™ Total RNA Isolation System (ThermoFisher Scientific). RNA quality and quantity were analyzed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA). Libraries were constructed using the Illumina TruSeq RNA Sample Prep Kit v2 and were sequenced on an Illumina HiSeq 2500 in a 100-cycle paired-end sequencing run (Illumina Inc., San Diego, CA, USA) at the Iowa State University DNA core facility. Sequence data were imported, quality trimmed in CLC Genomic workbench V 9.5.2, and mapped to the Meleagris gallopavo reference assembly 5.0 (17 (link)). Expression values were calculated only using uniquely mapped reads. Empirical analysis of differential gene expression was performed using the EdgeR statistical test, implemented in CLC Genomic workbench, on the raw unique reads (18 (link)). Gene expression differences greater than 1.5-fold with false discovery rate-adjusted P-values less than 0.05 were considered significant.