Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Blood RNA sequencing was performed on a cohort of adults attending the Emergency Department with suspected infection who had subsequently-confirmed viral, bacterial, COVID19 and healthy controls

ABSTRACT: The cohort comprised patients recruited between Sept 2014- June 2020. It includes patients with bacteremia and positive viral diagnostic test in the context of acute admission. All patients with definite infection were used for signature discovery. Whole blood was collected at the time of recruitment in Tempus Blood RNA tubes and total RNA was isolated with the Tempus Spin RNA Isolation Kit (ThermoFisher Scientific) according to the manufacturer’s instructions. RNA samples were stored at −80 °C until further analysis. After additional DNAse treatment, library preparation and sequencing of 30 million 150bp, paired end reads were conducted using the Illumina's TruSeq® RNA Sample Preparation Kit; ribosomal and globin RNA depletion was performed using the Illumina Ribo-Zero Gold kit and HiSeq 4000 at The Wellcome Centre for Human Genetics in Oxford UK.

INSTRUMENT(S): Illumina HiSeq 4000

ORGANISM(S): Homo sapiens

SUBMITTER: Myrsini Kaforou

PROVIDER: E-MTAB-13307 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The main aim of the study was to compare genome-wide gene expression profiles obtained from the two widely used commercially available whole blood RNA collection systems - PAXgeneTM and TempusTM tubes. Comparisons of present call rates, variances, correlations and influence of globin reduction across the two collection systems was performed using in vivo glucocorticoid stimulation in 24 peripheral blood samples from three individuals. Three healthy male volunteers aged 30-35 years with body mass index of 20-25 were enrolled in the study.1.5 mg dexamethasone stimulated peripheral blood samples were drawn 3 hours later. For each sample, duplicate measures of 2.5 ml and 3ml blood was collected in PAXgeneTM tubes (PreAnalytiX GmbH, Hombrechtikon, Switzerland) and TempusTM tubes (Applied Biosystems, Foster City, CA, USA) respectively. The PAXgeneTM and TempusTM tubes were stored for 2.5 hours at room temperature and then transferred to -20°C. Due to the lower RNA yield from the PAXgeneTM tubes, RNA from two PAXgeneTM tubes was pooled together for the experiment . Total RNA was isolated from whole blood stored in PAXgeneTM and TempusTM tubes according to the respective manufacturer's instructions. The PAXgeneTM samples were processed using the PAXgeneTM Blood RNA Kit based on the Quiagen method for column purification of nucleic acids (PreAnalytiX GmbH, Hombrechtikon, Switzerland, catalogue number 762174). The TempusTM samples were isolated using the TempusTM Spin RNA Isolation Kit (Applied Biosystems, Foster City, CA, USA, catalogue number 4380204). RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The concentration and purity of total RNA was independently assessed by 260/280 UV absorption ratios, respectively (Nanophotometer, Implen, Munich, Germany). RNA samples were divided into non-globin reduced and globin reduced groups. The GLOBINclearTM-Human Kit (Ambion, Inc., Texas, USA, catalogue number #AM1980) was used to remove globin mRNA. Sample amplification and labeling for both globin-reduced and non-reduced samples was performed using the IlluminaR TotalPrep RNA Amplification Kit (Ambion, Inc., Texas, USA, catalogue number AMIL1791). RNA quality, yield and numbers of detected transcripts from the two RNA collection systems was comparable, with no significant differences between the tube types. Globin reduction resulted in a significant increase in present call rates in PAXgeneTM and TempusTM tubes and significant decrease in gene expression variance in both RNA collection tubes . Comparisons of glucocorticoid receptor-stimulated gene expression profiles between the two collection tube systems revealed an overlap of only 17 to 54%, depending on the stringency level of the statistical thresholds. This overlap increased by 1-8% when the RNA samples were processed to remove the globin mRNA. These results indicate that gene expression profiles obtained from PAXgeneTM and TempusTM differ drastically and should not be analyzed together. These data suggest that researchers must exert caution while interpreting expression profiles obtained through different RNA collection tubes.

Dataset Information

Blood RNA sequencing was performed on a cohort of adults attending the Emergency Department with suspected infection who had subsequently-confirmed viral, bacterial, COVID19 and healthy controls

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