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

2.50
Hdl Handle:
http://hdl.handle.net/10541/71919
Title:
Successful downstream application of the Paxgene Blood RNA system from small blood samples in paediatric patients for quantitative PCR analysis
Authors:
Carrol, Enitan D; Salway, Fiona; Pepper, Stuart D; Saunders, Emma K; Mankhambo, Limangeni A; Ollier, William E; Hart, C Anthony; Day, Phillip
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 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.
Affiliation:
Malawi-Liverpool-Wellcome Trust Clinical Research Programme, PO Box 30096, Blantyre, Malawi, Africa. edcarrol@liv.ac.uk
Citation:
Successful downstream application of the Paxgene Blood RNA system from small blood samples in paediatric patients for quantitative PCR analysis. 2007, 8:20 BMC Immunol.
Journal:
BMC immunology
Issue Date:
2007
URI:
http://hdl.handle.net/10541/71919
DOI:
10.1186/1471-2172-8-20
PubMed ID:
17850649
Type:
Article
Language:
en
ISSN:
1471-2172
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
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.en
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

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