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


CONCLUSIONS
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.
Carrol
Enitan D
ED
Malawi-Liverpool-Wellcome Trust Clinical Research Programme, PO Box 30096, Blantyre, Malawi, Africa. edcarrol@liv.ac.uk
Salway
Fiona
F
Pepper
Stuart D
SD
Saunders
Emma
E
Mankhambo
Limangeni A
LA
Ollier
William E
WE
Hart
C Anthony
CA
Day
Phillip
P
D000293:Adolescent; D001800:Blood Specimen Collection / Q000379:methods*; D016022:Case-Control Studies; D002648:Child; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D007223:Infant; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D008297:Male; D011008:Pneumococcal Infections / Q000097:blood; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000097:blood*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011933:Reagent Kits, Diagnostic
D015850:Interleukin-6; D012333:RNA, Messenger; D011933:Reagent Kits, Diagnostic; D018799:Intercellular Adhesion Molecule-1; D012313:RNA