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Successful downstream application of the Paxgene Blood RNA system from small blood samples in paediatric patients for quantitative PCR analysis

Carrol, Enitan D.; Salway, Fiona; Pepper, Stuart D.; Saunders, Emma; Mankhambo, Limangeni A.; Ollier, William E.; Hart, C. Anthony and Day, Phillip (2007) Successful downstream application of the Paxgene Blood RNA system from small blood samples in paediatric patients for quantitative PCR analysis. BMC Immunology, 8 . Article Number: 20. ISSN 1471-2172

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Abstract

Background: The challenge of gene expression studies is to reliably quantify levels of transcripts, but this is hindered by a number of factors including sample availability, handling and storage. The PAXgene™ Blood RNA System includes a stabilizing additive in a plastic evacuated tube, but requires 2.5 mL blood, which makes routine implementation impractical for paediatric use. The aim of this study was to modify the PAXgene™ Blood RNA System kit protocol for application to small, sick chidren, without compromising RNA integrity, and subsequently to perform quantitative analysis of ICAM and interleukin-6 gene expression. Aliquots of 0.86 mL PAXgene™ reagent were put into microtubes and 0.3 mL whole blood added to maintain the same recommended proportions as in the PAXgene™ evacuated tube system. RNA quality was assessed using the Agilent BioAnalyser 2100 and an in-house TaqMan™ assay which measures GAPDH transcript integrity by determining 3' to 5' ratios. qPCR analysis was performed on an additional panel of 7 housekeeping genes. Three reference genes (HPRT1, YWHAZ and GAPDH) were identified using the GeNORM algorithm, which were subsequently used to normalising target gene expression levels. ICAM-1 and IL-6 gene expression were measured in 87 Malawian children with invasive pneumococcal disease. Results: Total RNA yield was between 1,114 and 2,950 ng and the BioAnalyser 2100 demonstrated discernible 18s and 28s bands. The cycle threshold values obtained for the seven housekeeping genes were between 15 and 30 and showed good consistency. Median relative ICAM and IL-6 gene expression were significantly reduced in non-survivors compared to survivors (ICAM: 3.56 vs 4.41, p = 0.04, and IL-6: 2.16 vs 6.73, p = 0.02). Conclusion: We have successfully modified the PAXgene™ blood collection system for use in small children and demonstrated preservation of RNA integrity and successful quantitative real-time PCR analysis.

Item Type:Article
Additional Information:Published: 12 September 2007. 8 pages (page numbers not for citation purposes).
Uncontrolled Keywords:INTERCELLULAR-ADHESION MOLECULE-1; GENE-EXPRESSION; MESSENGER-RNA; WHOLE-BLOOD; STREPTOCOCCUS-PNEUMONIAE; DIFFERENTIAL EXPRESSION; HEALTHY-SUBJECTS; POLYMORPHISM; MENINGITIS; BARRIER
Subjects:R Medicine > R Medicine (General)
R Medicine > RA Public aspects of medicine
R Medicine > RJ Pediatrics > RJ101 Child Health. Child health services
Departments, Research Centres and Related Units:Academic Faculties, Institutes and Research Centres > Faculty of Medicine > School of Infection & Host Defence
Academic Faculties, Institutes and Research Centres > Faculty of Medicine > School of Reproductive & Developmental Medicine
DOI:10.1186/1471-2172-8-20
Publisher's Statement:© 2007 Carrol et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Related URLs:
Refereed:Yes
Status:Published
ID Code:685
Deposited On:25 Jun 2008 11:53
Last Modified:28 Feb 2012 12:07

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