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Transimmunom whole blood RNA-seq data from type 1 diabetic patients and healthy volunteers

ABSTRACT: Patients affected by type 1 diabetes are recruited in the departments of Diabetology and Healthy Volunteers (HV) are selected based on internal records in the same hospital. Total RNA from whole blood has been extracted following a two-step procedure. First, RNA from blood collected on PAX-Gene tubes has been extracted using Maxwell 16 LEV simplyRNA blood kit (Promega) following manufacturer recommendations and second b-globin, dominant RNA from red blood cells, has been removed using the GLOBINclear kit (Ambion) on extracted RNA. RNA sequencing has been performed from using the TruSeq Stranded mRNA preparation kit (Illumina) on 500 ng b-globin depleted RNA with a RNA Integrity Number > 8 (measured on Bioanalyzer following manufacturer recommendations), and then sequenced following a pair-end 2x75 bp protocol on NextSeq 500 or HiSeq 4000 (Illumina) at LIGAN Equipex (Lille, France).

ORGANISM(S): Homo sapiens

PROVIDER: GSE123658 | GEO | 2020/06/01

REPOSITORIES: GEO

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Project description:Whole blood rather than purified peripheral blood mononuclear cells is likely to become the prime tissue using expression microarrays for disease predication or prognosis however excess of globin mRNA may reduce probe detection sensitivity. In our study, we assessed whether whole-blood or globin-reduced RNA gives the most robust and sensitive results to detect small gene expression changes in response to hormone replacement therapy exposure. Each sample (N = 12) were hybridized according to 3 different protocols: no globin reduction (controls), globin reduction using peptid nucleic acids (PNA) and using magnetic beads in the GlobinClear kit from Ambion. Finally, 7 and 4 technical replicates were conducted in no globin reduction and PNA groups, respectively. Both globin reduction approaches were mostly efficient at reducing globin RNA from cRNA. Samples processed by GlobinClear kit gave a very distinct gene expression profiles from the controls while samples processed with PNA gave an intermediary profile closest to the non globin reduction group with a slight increased sensitivity of transcript detection but a loss of reproducibility. Overall, no sign of higher sensitivity in detection of gene expression changes followed by hormone exposure was observed after globin reduction which was therefore judged not beneficial. Keywords: Groups comparaison To assess effect of globin reduction protocols on gene expression from whole-blood, we selected 12 postmenopausal women (6 HRT users and 6 non-HRT users) who were not using other medication than HRT at the time of blood sampling and in order to cover a wide body mass index (BMI) range in both HRT and non HRT users. The samples were also selected to contain the highest concentration of total extracted RNA. Each sample was hybridized according to 3 different protocols: no globin reduction (controls), globin reduction using PNAs, globin reduction using magnetic beads in the GlobinClear™ kit from Ambion. The reproducibility of the globin reduction method compared to the non globin reduction approach was investigated for the PNA method only since this method was the most effective and specific. We planned to conduct 7 technical replicates in each group (i.e. no globin reduction and PNA groups). During amplification process with PNA, 4 samples failed to reverse transcript long fragment of mRNA certainly due to inhibitory contamination and these arrays were therefore excluded from our analyses. One sample (sample 34) was amplified with PNA twice the same day to study technical reproducibility without amplification date effect. Finally, a total of 47 arrays were conducted including 7 and 4 technical replicates in no globin reduction and PNA group, respectively.

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Transimmunom whole blood RNA-seq data from type 1 diabetic patients and healthy volunteers

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