Isolation of Microarray-Grade Total RNA, MicroRNA, and DNA from a Single PAXgene Blood RNA Tube

Kruhøffer, Mogens; Dyrskjøt, Lars; Voß, Thorsten; Lindberg, Raija L. P.; Wyrich, Ralf; Thykjær, Thomas; Ørntoft, Torben F.

doi:10.2353/jmoldx.2007.060175

Cited by 43 publications

(33 citation statements)

References 30 publications

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“…In this context, it is worth noting that we were able to obtain technically robust and informative microarray analyses from a single 2.5 ml blood sample in 56/57 (98.2%) cases. These results expand recent studies showing that extraction of microarray-grade RNA from frozen whole blood stabilized in PAXGene tubes is technically feasible 23,25 to the setting of a European multicenter phase I trial including non-academic study sites. There were two important clinical observations in this study that we aimed to further investigate by our microarray analysis.…”

Section: Pharmacodynamic Effects Of Spc2996 In Cll Patientssupporting

confidence: 74%

The novel antisense Bcl-2 inhibitor SPC2996 causes rapid leukemic cell clearance and immune activation in chronic lymphocytic leukemia

et al. 2011

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SPC2996 is a novel locked nucleic acid phosphorothioate antisense molecule targeting the mRNA of the Bcl-2 oncoprotein. We investigated the mechanism of action of SPC2996 and the basis for its clinically observed immunostimulatory effects in chronic lymphocytic leukemia (CLL). Patients with relapsed CLL were treated with a maximum of six doses of SPC2996 (0.2-6 mg/kg) in a multicenter phase I trial. Microarraybased transcriptional profiling of circulating CLL cells was carried out before and after the first infusion of SPC2996 in 18 patients. Statistically significant transcriptomic changes were observed at doses X4 mg/kg and occurred as early as 24 h after the first infusion of the oligonucleotide. SPC2996 induced the upregulation of 466 genes including a large number of immune response and apoptotic regulator molecules, which were enriched for Toll-like receptor response genes. Serum measurements confirmed the release of pro-inflammatory cytokines including chemokine (C-C motif) ligand 3 (macrophage inflammatory protein 1a) and tumor necrosis factor-a, thereby validating the in vivo transcriptomic data at the protein level. SPC2996 caused a X50% reduction of circulating lymphocytes in five of 18 (28%) patients, which was found to be independent of its immunostimulatory and anti-Bcl-2 effects.

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Section: Pharmacodynamic Effects Of Spc2996 In Cll Patientssupporting

confidence: 74%

The novel antisense Bcl-2 inhibitor SPC2996 causes rapid leukemic cell clearance and immune activation in chronic lymphocytic leukemia

et al. 2011

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“…In general, miRNA species are much more stable than mRNA and most studies have not used these methods. Several studies have demonstrated successful isolation and quantitation of specific miRNAs or whole miRNomes from whole blood [42][43][44][45] or tissue [46]. However, few studies have systematically compared RNA stabilization media with a control method.…”

Section: Rna Preservation Reagentsmentioning

confidence: 99%

Variability in, variability out: best practice recommendations to standardize pre-analytical variables in the detection of circulating and tissue microRNAs

Khan

Lieberman

Lockwood

2017

Clinical Chemistry and Laboratory Medicine (CCLM)

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microRNAs (miRNAs) hold promise as biomarkers for a variety of disease processes and for determining cell differentiation. These short RNA species are robust, survive harsh treatment and storage conditions and may be extracted from blood and tissue. Pre-analytical variables are critical confounders in the analysis of miRNAs: we elucidate these and identify best practices for minimizing sample variation in blood and tissue specimens. Preanalytical variables addressed include patient-intrinsic variation, time and temperature from sample collection to storage or processing, processing methods, contamination by cells and blood components, RNA extraction method, normalization, and storage time/conditions. For circulating miRNAs, hemolysis and blood cell contamination significantly affect profiles; samples should be processed within 2 h of collection; ethylene diamine tetraacetic acid (EDTA) is preferred while heparin should be avoided; samples should be "double spun" or filtered; room temperature or 4 °C storage for up to 24 h is preferred; miRNAs are stable for at least 1 year at -20 °C or -80 °C. For tissuebased analysis, warm ischemic time should be < 1 h; cold ischemic time (4 °C) < 24 h; common fixative used for all specimens; formalin fix up to 72 h prior to processing; enrich for cells of interest; validate candidate biomarkers with in situ visualization. Most importantly, all specimen types should have standard and common workflows with careful documentation of relevant pre-analytical variables.

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“…After the contents were sufficiently mixed, tubes were placed for 2 h at room temperature and then stored at 2 808C. Although the stability of RNA in PAXgene tubes is guaranteed up to 2.5 years (Kruhoffer et al 2007), total RNA was extracted within 1 month using a PAXgene Blood RNA kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The total RNA yield was measured by NanoDrop-1000 (Nanodrop, Wilmington, DE, USA).…”

Section: Peripheral Blood Lymphocytes Rna and Cdna Preparationmentioning

confidence: 99%

Effects of chronic academic stress on mental state and expression ofglucocorticoid receptor αand β isoforms in healthy Japanese medical students

Kurokawa

Tanahashi

Murata

et al. 2011

Stress

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Chronic academic stress responses were assessed by measuring mental state, salivary cortisol levels, and the glucocorticoid receptor (GR) gene expression in healthy Japanese medical students challenging the national medical license examination. Mental states of 17 male and 9 female medical undergraduates, aged 25.0 ± 1.2 years (mean ± SD), were assessed by the State and Trait Anxiety Inventory (STAI) and the Self-Rating Depression Scale (SDS) 2 months before, 2 days before, and 1 month after the examination. At the same time points, saliva and blood were collected. STAI-state scores peaked 2 days before the examination. Scores on STAI-trait and SDS, and salivary cortisol levels were consistently higher during the pre-examination period. One month after the examination, all these measures had significantly decreased to baseline levels. Real-time reverse transcription PCR showed that this chronic anxious state did not change the expression of the functional GRα mRNA isoform in peripheral leukocytes, while it resulted in reduced expression of the GRβ isoform 2 days before the examination. Our results replicate and extend a significant impact of chronic academic stressors on the mental state of healthy Japanese medical students and suggest a possible association of GRβ gene in response to psychological stress.

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Isolation of Microarray-Grade Total RNA, MicroRNA, and DNA from a Single PAXgene Blood RNA Tube

Cited by 43 publications

References 30 publications

The novel antisense Bcl-2 inhibitor SPC2996 causes rapid leukemic cell clearance and immune activation in chronic lymphocytic leukemia

The novel antisense Bcl-2 inhibitor SPC2996 causes rapid leukemic cell clearance and immune activation in chronic lymphocytic leukemia

Variability in, variability out: best practice recommendations to standardize pre-analytical variables in the detection of circulating and tissue microRNAs

Effects of chronic academic stress on mental state and expression ofglucocorticoid receptor αand β isoforms in healthy Japanese medical students

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