Evaluation of a low-cost procedure for sampling, long-term storage, and extraction of RNA from blood for qPCR analyses
-
Rasmus B. Mærkedahl
,
Hanne Frøkiær
Abstract
Background: In large clinical trials, where RNA cannot be extracted immediately after sampling, preserving RNA in whole blood is a crucial initial step in obtaining robust qPCR data. The current golden standard for RNA preservation is costly and designed for time-consuming column-based RNA-extraction. We investigated the use of lysis buffer for long-term storage of blood samples for qPCR analysis.
Methods: Blood was collected from 13 healthy adults and diluted in MagMAX lysis/binding solution or PAXgene Blood RNA tubes and stored at –20 °C for 0, 1, or 4 months before RNA extraction by the matching method. RNA integrity, yield and purity were evaluated and the methods were compared by subsequent analyses of the gene expression levels of 18S, ACTB, IL1B, IL1RN, IL1R2, and PGK1 using qPCR.
Results: The MagMAX system extracted 2.3–2.8 times more RNA per mL blood, with better performance in terms of purity, and with comparable levels of integrity relative to the PAXgene system. Gene expression analysis using qPCR of 18S, ACTB, IL1B, IL1RN, IL1R2, and the promising blood-specific reference gene, PGK1, revealed negligible differences (<1-fold) between the samples stored in MagMAX lysis/binding solution over time and between samples stored and extracted by the two systems.
Conclusions: The MagMAX system can be used for storage of human blood for up to 4 months and is equivalent to the PAXgene system for RNA extraction. It furthermore, provides a means for significant cost reduction in large clinical trials.
Acknowledgments
We would like to thank Professor Lars Ove Dragsted for providing us with PAXgene blood RNA tubes and the Gut, Grain & Greens (3G) center for providing us with samples for these analyses. We would also like to thank Anne Friis Petersen for letting us use her Bioanalyzer, and Anita Pacht for guidance in its use. Finally we would like to thank the Danish Council for Strategic Research for funding this project (grant number 11-116163).
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Financial support: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
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Supplemental Material
The online version of this article (DOI: 10.1515/cclm-2014-1054) offers supplementary material, available to authorized users.
©2015 by De Gruyter
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Artikel in diesem Heft
- Frontmatter
- Editorials
- Once upon a time: a tale of ISO 15189 accreditation
- A new integrated tool for assessing and monitoring test comparability and stability
- Liver-FibroSTARD checklist and glossary: tools for standardized design and reporting of diagnostic accuracy studies of liver fibrosis tests
- Reviews
- Thromboembolic risk in hematological malignancies
- A review of the cut-off points for the diagnosis of vitamin B12 deficiency in the general population
- Opinion Paper
- Permissible limits for uncertainty of measurement in laboratory medicine
- EFLM Position Paper
- Flexible scope for ISO 15189 accreditation: a guidance prepared by the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group Accreditation and ISO/CEN standards (WG-A/ISO)
- Genetics and Molecular Diagnostics
- Evaluation of a low-cost procedure for sampling, long-term storage, and extraction of RNA from blood for qPCR analyses
- Application of real-time PCR of sex-independent insertion-deletion polymorphisms to determine fetal sex using cell-free fetal DNA from maternal plasma
- General Clinical Chemistry and Laboratory Medicine
- The Empower project – a new way of assessing and monitoring test comparability and stability
- Comparison of four automated serum vitamin B12 assays
- Combined indicator of vitamin B12 status: modification for missing biomarkers and folate status and recommendations for revised cut-points
- INR vs. thrombin generation assays for guiding VKA reversal: a retrospective comparison
- Determination of dabigatran in plasma, serum, and urine samples: comparison of six methods
- Simple high-throughput analytical method using ultra-performance liquid chromatography coupled with tandem mass spectrometry to quantify total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in urine
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- Relationship between antiphosphatidylserine/prothrombin and conventional antiphospholipid antibodies in primary antiphospholipid syndrome
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- Relevance of EDTA carryover during blood collection
- Reference intervals for renal injury biomarkers neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 in young infants
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