BACKGROUND: Current gene expression analysis relies on the assumption that the isolated RNA represents all species of mRNA in proportions equal to those in the original materials. No system is available for absolute quantification of mRNA. METHODS: We applied whole blood to 96-well filterplates to trap leukocytes. Lysis buffer containing cocktails of specific reverse primers and known concentrations of synthetic external control RNA (RNA34) was added to filterplates, and cell lysates were transferred to oligo(dT)-immobilized microplates for hybridization. We then synthesized the cDNA in the oligo(dT)-immobilized microplates from these primer sites and used the cDNA for real-time PCR. RNA34 acted as a universal control, and gene amplification results were converted to quantities of mRNA per microliter of whole blood after the recovery of RNA34 in each sample was determined. RESULTS: Under fully optimized conditions, both added RNA34 and native mRNA species exhibited approximately 10% recovery from whole blood to real-time PCR. When whole blood was stimulated ex vivo, changes in gene expression as low as 30%-40% were detected with statistical significance, and the experimental CVs were low (10%-20%). CONCLUSION: This new system to estimate mRNA copies per microliter of whole blood may allow standardization of gene-expression-based molecular diagnostics.
BACKGROUND: Current gene expression analysis relies on the assumption that the isolated RNA represents all species of mRNA in proportions equal to those in the original materials. No system is available for absolute quantification of mRNA. METHODS: We applied whole blood to 96-well filterplates to trap leukocytes. Lysis buffer containing cocktails of specific reverse primers and known concentrations of synthetic external control RNA (RNA34) was added to filterplates, and cell lysates were transferred to oligo(dT)-immobilized microplates for hybridization. We then synthesized the cDNA in the oligo(dT)-immobilized microplates from these primer sites and used the cDNA for real-time PCR. RNA34 acted as a universal control, and gene amplification results were converted to quantities of mRNA per microliter of whole blood after the recovery of RNA34 in each sample was determined. RESULTS: Under fully optimized conditions, both added RNA34 and native mRNA species exhibited approximately 10% recovery from whole blood to real-time PCR. When whole blood was stimulated ex vivo, changes in gene expression as low as 30%-40% were detected with statistical significance, and the experimental CVs were low (10%-20%). CONCLUSION: This new system to estimate mRNA copies per microliter of whole blood may allow standardization of gene-expression-based molecular diagnostics.
Authors: K Omori; M Mitsuhashi; K Ishiyama; I Nair; J Rawson; I Todorov; F Kandeel; Y Mullen Journal: Diabetologia Date: 2011-05-13 Impact factor: 10.122
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