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

University of California, Irvine, Irvine, California, United States
Clinical Chemistry (Impact Factor: 7.91). 05/2006; 52(4):634-42. DOI: 10.1373/clinchem.2005.048983
Source: PubMed


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.

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    • "Eighty µL of Lysis buffer [14] were added to the filters, and incubated at 37°C for 10 min. The lysates were then transferred to an oligo(dT)-immobilized microplate (HCR) by centrifugation and incubated at 4°C for overnight for mRNA hybridization [14]. After six washes with Wash buffers, cDNA was synthesized in the same microplate by adding 30 µL of 1× reverse transcription buffer containing 1.25 mM each of dNTPs, 2.7 U/µL MMLV reverse transcriptase and 0.13 U/µL RNasin, and incubated at 37°C for 2 hours. "
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    • "Rat spleen mRNA and human spleen mRNA were from Clontech Laboratories. Preparation of in vitro transcribed CD4 mRNA (742 bases long) was described elsewhere (18). "
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