Stabilization of mRNA Expression in Whole Blood Samples

Rainen, Lynne; Oelmueller, Uwe; Jurgensen, Stewart; Wyrich, Ralf; Ballas, Cynthia; Schram, Jim; Herdman, Chris M.; Bankaitis-Davis, Danute; Nicholls, N; Trollinger, David; Tryon, Victor V.

doi:10.1093/clinchem/48.11.1883

Cited by 294 publications

(153 citation statements)

References 22 publications

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“…Thus, storage of blood in PAXgene TM for up to 5 days did not affect R1 or R2 expression when normalized to that of b-actin. This observation supports the manufacturer's claim that RNA is stabilized for several days at room temperature (9), and extends the results by demonstrating reproducible downstream application of the isolated RNA.…”

Section: R1 R2 and B-actin Gene Expression In Extracted Rna Samplessupporting

confidence: 88%

Optimization of the PAXgene™ blood RNA extraction system for gene expression analysis of clinical samples

Chai¹,

Vassilakos²,

Lee³

et al. 2005

J. Clin. Lab. Anal.

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One major problem associated with collecting whole blood from patients for use as a source of RNA in gene expression studies is that the RNA degrades during collection and storage. Preservation of RNA quality is vital in such studies because the stability of the RNA ultimately affects analysis of gene expression. In this study the PAXgene blood collection system was compared with a standard erythrocyte lysis method for isolating RNA from blood samples. The methods were compared in terms of RNA yield, RNA stabilization, and DNA contamination. The study also included the downstream application to RT-PCR analysis for relative mRNA expression levels of the ribonucleotide reductase subunits R1 and R2. The results show that blood collection in conventional collection tubes, and leukocyte isolation by erythrocyte lysis lead to significant degradation of RNA. Our findings confirm the ability of PAXgene to stabilize RNA in whole blood; however, RNA extracted by the PAXgene method contained significant DNA contamination. Given the low basal expression of the target genes analyzed in this study, contaminating DNA could potentially affect accurate interpretation of RT-PCR data. As a result, the PAXgene protocol was optimized to include off-column DNase treatments, which yielded high-quality RNA suitable for gene expression studies. Furthermore, the results suggest that RNA isolation with PAXgene is advantageous compared to traditional extraction methods for RT-PCR analysis of large or different-sized amplicons.

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Section: R1 R2 and B-actin Gene Expression In Extracted Rna Samplessupporting

confidence: 88%

Optimization of the PAXgene™ blood RNA extraction system for gene expression analysis of clinical samples

Chai¹,

Vassilakos²,

Lee³

et al. 2005

J. Clin. Lab. Anal.

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“…preservation time 24 h, Schwochow et al 2012; Table 2) and in expression studies (i.e. preservation time 5 days, Rainen et al 2002; Table 2).…”

Section: Rna Preservationmentioning

confidence: 99%

Preservation of RNA and DNA from mammal samples under field conditions

Camacho‐Sanchez

Burraco

Gómez-Mestre

et al. 2013

Molecular Ecology Resources

157

138

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Ecological and conservation genetics require sampling of organisms in the wild. Appropriate preservation of the collected samples, usually by cryostorage, is key to the quality of the genetic data obtained. Nevertheless, cryopreservation in the field to ensure RNA and DNA stability is not always possible. We compared several nucleic acid preservation solutions appropriate for field sampling and tested them on rat (Rattus rattus) blood, ear and tail tip, liver, brain and muscle. We compared the efficacy of a nucleic acid preservation (NAP) buffer for DNA preservation against 95% ethanol and Longmire buffer, and for RNA preservation against RNAlater (Qiagen) and Longmire buffer, under simulated field conditions. For DNA, the NAP buffer was slightly better than cryopreservation or 95% ethanol, but high molecular weight DNA was preserved in all conditions. The NAP buffer preserved RNA as well as RNAlater. Liver yielded the best RNA and DNA quantity and quality; thus, liver should be the tissue preferentially collected from euthanized animals. We also show that DNA persists in nonpreserved muscle tissue for at least 1 week at ambient temperature, although degradation is noticeable in a matter of hours. When cryopreservation is not possible, the NAP buffer is an economical alternative for RNA preservation at ambient temperature for at least 2 months and DNA preservation for at least 10 months.

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“…We were interested in using blood as a snapshot of the ongoing lung infection and potentially as an indicator of disease progression in the animal as a whole. Our study included the use of blood collection tubes (PAXgene blood RNA system) that stabilize RNA in whole blood, so the effects of handling and storage on active transcripts were minimized (11,16,61). In this manner, we were able to study gene expression in all white blood cells as opposed to only peripheral mononuclear cells.…”

Section: Progression and Patterns Of Global Gene Expression From Daysmentioning

confidence: 99%

Integrated Molecular Signature of Disease: Analysis of Influenza Virus-Infected Macaques through Functional Genomics and Proteomics

Baas¹,

Baskin

Diamond³

et al. 2006

J Virol

107

112

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Recent outbreaks of avian influenza in humans have stressed the need for an improved nonhuman primate model of influenza pathogenesis. In order to further develop a macaque model, we expanded our previous in vivo genomics experiments with influenza virus-infected macaques by focusing on the innate immune response at day 2 postinoculation and on gene expression in affected lung tissue with viral genetic material present. Finally, we sought to identify signature genes for early infection in whole blood. For these purposes, we infected six pigtailed macaques (Macaca nemestrina) with reconstructed influenza A/Texas/36/91 virus and three control animals with a sham inoculate. We sacrificed one control and two experimental animals at days 2, 4, and 7 postinfection. Lung tissue was harvested for pathology, gene expression profiling, and proteomics. Blood was collected for genomics every other day from each animal until the experimental endpoint. Gross and microscopic pathology, immunohistochemistry, viral gene expression by arrays, and/or quantitative real-time reverse transcription-PCR confirmed successful yet mild infections in all experimental animals. Genomic experiments were performed using macaque-specific oligonucleotide arrays, and high-throughput proteomics revealed the host response to infection at the mRNA and protein levels. Our data showed dramatic differences in gene expression within regions in influenza virus-induced lesions based on the presence or absence of viral mRNA. We also identified genes tightly coregulated in peripheral white blood cells and in lung tissue at day 2 postinoculation. This latter finding opens the possibility of using gene expression arrays on whole blood to detect infection after exposure but prior to onset of symptoms or shedding.

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Stabilization of mRNA Expression in Whole Blood Samples

Cited by 294 publications

References 22 publications

Optimization of the PAXgene™ blood RNA extraction system for gene expression analysis of clinical samples

Optimization of the PAXgene™ blood RNA extraction system for gene expression analysis of clinical samples

Preservation of RNA and DNA from mammal samples under field conditions

Integrated Molecular Signature of Disease: Analysis of Influenza Virus-Infected Macaques through Functional Genomics and Proteomics

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