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dc.contributor.authorCarrol, Enitan D
dc.contributor.authorSalway, Fiona
dc.contributor.authorPepper, Stuart D
dc.contributor.authorSaunders, Emma K
dc.contributor.authorMankhambo, Limangeni A
dc.contributor.authorOllier, William E
dc.contributor.authorHart, C Anthony
dc.contributor.authorDay, Phillip
dc.date.accessioned2009-06-30T11:10:42Z
dc.date.available2009-06-30T11:10:42Z
dc.date.issued2007
dc.identifier.citationSuccessful downstream application of the Paxgene Blood RNA system from small blood samples in paediatric patients for quantitative PCR analysis. 2007, 8:20 BMC Immunol.en
dc.identifier.issn1471-2172
dc.identifier.pmid17850649
dc.identifier.doi10.1186/1471-2172-8-20
dc.identifier.urihttp://hdl.handle.net/10541/71919
dc.description.abstractBACKGROUND: 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 children, 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.
dc.language.isoenen
dc.subject.meshAdolescent
dc.subject.meshBlood Specimen Collection
dc.subject.meshCase-Control Studies
dc.subject.meshChild
dc.subject.meshFemale
dc.subject.meshGene Expression Regulation
dc.subject.meshHumans
dc.subject.meshInfant
dc.subject.meshIntercellular Adhesion Molecule-1
dc.subject.meshInterleukin-6
dc.subject.meshMale
dc.subject.meshPneumococcal Infections
dc.subject.meshPolymerase Chain Reaction
dc.subject.meshRNA
dc.subject.meshRNA, Messenger
dc.subject.meshReagent Kits, Diagnostic
dc.titleSuccessful downstream application of the Paxgene Blood RNA system from small blood samples in paediatric patients for quantitative PCR analysisen
dc.typeArticleen
dc.contributor.departmentMalawi-Liverpool-Wellcome Trust Clinical Research Programme, PO Box 30096, Blantyre, Malawi, Africa. edcarrol@liv.ac.uken
dc.identifier.journalBMC immunologyen
html.description.abstractBACKGROUND: 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 children, 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.


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