Quantification of mRNA in Whole Blood by Assessing Recovery of RNA and Efficiency of cDNA Synthesis

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. We applied whole blood to 96-well filterplates to trap leukocytes. Lysis...

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Bibliographic Details
Published in:Clinical chemistry (Baltimore, Md.) Md.), 2006-04, Vol.52 (4), p.634-642
Main Authors: Mitsuhashi, Masato, Tomozawa, Shigeru, Endo, Katsuya, Shinagawa, Atsushi
Format: Article
Language:English
Subjects:
Adult
Analytical, structural and metabolic biochemistry
Biological and medical sciences
Cyclin-Dependent Kinase Inhibitor p21 - blood
Cyclin-Dependent Kinase Inhibitor p21 - genetics
DNA, Complementary - chemical synthesis
Efficiency
Fundamental and applied biological sciences. Psychology
Gene amplification
Gene Expression
Humans
Investigative techniques, diagnostic techniques (general aspects)
Leukocytes
Leukocytes - metabolism
Medical sciences
Poly A - blood
Reference Standards
Reverse Transcriptase Polymerase Chain Reaction - standards
RNA - blood
RNA, Messenger - blood
Standardization
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Summary: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. 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. 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%). This new system to estimate mRNA copies per microliter of whole blood may allow standardization of gene-expression-based molecular diagnostics.
ISSN:0009-9147
1530-8561
DOI:10.1373/clinchem.2005.048983