Profiling Gene Expression in Whole Blood Samples Following an In-Vitro Challenge

Spijker, Sabine; Leemput, Joyce van de; Hoekstra, Chantal; Boomsma, Dorret I.; Smit, August B.

doi:10.1375/1369052042663878

Cited by 16 publications

(13 citation statements)

References 21 publications

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“…In terms of RNA quality and gene expression, however, our results from BM samples show excellent pairwise correlation between different incubation times using quantitative RT-PCR and are in line with those derived from PB (3,26,27 ). The PAXgene system is appropriate for quantification of gene expression levels in BM samples.…”

Section: Discussionsupporting

confidence: 81%

Preanalytical mRNA Stabilization of Whole Bone Marrow Samples

et al. 2007

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Background: Gene expression profiling is a useful tool for cancer diagnosis and basic research. A major limitation is that, even during short-term storage of native specimens of peripheral blood or bone marrow (BM) and/or RNA isolation, significant changes of gene expression pattern can occur because of gene induction, repression, and RNA degradation. Methods: We investigated the effectiveness of a newly developed RNA stabilization and preparation system for BM specimens (PAXgene™ Bone Marrow RNA System) over time. We analyzed 256 RNA samples, processed from 64 BM specimens. Results: Although the overall RNA yield (normalized to 1 ؋ 10 7 leukocytes) was not different, the RNA preparation using unstabilized reference samples had an ϳ3 times higher failure rate. With the PAXgene system, we observed significantly higher RNA integrity compared with the reference RNA preparation system (P <0.01). In the stabilized samples, we found very high pairwise correlation in gene expression (⌬⌬C T 0.16 -0.53) for the analyzed genes (GATA1, RUNX1, NCAM1, and SPI1) after 48-h storage compared with immediate preparation of RNA (2 h after BM collection). However, we found major differences in half of the analyzed genes using the reference RNA isolation procedure (⌬⌬C T 1.07 and 1.32). Conclusions: The PAXgene system is able to stabilize RNA from clinical BM samples and is suitable to isolate high-quality and -quantity RNA.

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Section: Discussionsupporting

confidence: 81%

Preanalytical mRNA Stabilization of Whole Bone Marrow Samples

et al. 2007

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“…When the home visit takes place, six tubes of blood (EDTA, heparin, serum, citrate) and two small tubes of urine are collected. RNA samples are collected in challenged and unchallenged whole blood samples (Spijker et al, 2004). A few traits (e.g., lipids, CRP, insulin, glucose, white blood cell count, HbA1c) are assessed immediately in fresh blood samples, and the rest of the material is frozen for later processing.…”

Section: Twin Research and Human Genetics December 2006mentioning

confidence: 99%

Netherlands Twin Register: From Twins to Twin Families

Boomsma¹,

et al. 2006

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“…However, the main benefit of ex vivo RNA stabilization is the possibility of fluently transportation of unfrozen sample material. Furthermore, immediate snap-freezing of anticoagulated whole blood was shown to be a reasonable alternative to more expensive blood collection systems that include RNA stabilizing additives [38]. Increased TF transcription rates in circulating monocytes have been associated with a hypercoagulable state [5][6][7]9].…”

Section: Discussionmentioning

confidence: 99%

Tissue Factor Gene Expression Analysis in Circulating Monocytes*

Müller¹,

Rox²,

Pötzsch³

2006

Transfus Med Hemother

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Background: There is growing evidence that an increase in stimulated and tissue factor(TF)-expressing monocytes drive the coagulation system to a hypercoagulable state. In order to further investigate this phenomenon, methods are needed that allow sensitive, reliable and specific quantification of TF-expressing monocytes. Materials and Methods: Two procedures of one-step multiplex real-time RT-PCR were developed that allow absolute quantification of TF, CD14 and glyceraldehyde-3- phosphate dehydrogenase (GAPDH) mRNA transcripts. In order to minimize preanalytical changes of the mRNA profile, a blood sampling system which is based on integrated RNA stabilization was used. Results: An in vitro lipopolysaccharide (LPS) model was used for initial assay evaluation. LPS-induced TF gene expression patterns were similar to previously described findings. With the use of the whole blood RNA stabilization system, the marker transcripts were preserved for up to 24 h at room temperature and for up to 5 days at 4 °C. Monocytic TF transcription analysis in different patient groups identified significant higher levels in thrombophilic patients than in a representative control population. In all patients and control probands analyzed we found significantly higher levels of TF transcripts in carriers of the G allele of the TF-603A/G promotor polymorphism. Conclusion: The newly developed RT-PCR allows sensitive, reliable and specific quantification of TF transcripts in whole blood samples.

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Profiling Gene Expression in Whole Blood Samples Following an In-Vitro Challenge

Cited by 16 publications

References 21 publications

Preanalytical mRNA Stabilization of Whole Bone Marrow Samples

Preanalytical mRNA Stabilization of Whole Bone Marrow Samples

Netherlands Twin Register: From Twins to Twin Families

Tissue Factor Gene Expression Analysis in Circulating Monocytes*

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