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

Abstract

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 ϳ10% 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.

Quantification of Drug-Induced mRNA in Human Whole Blood ex vivo

“…The cyclin-dependent kinase inhibitor 1A (p21) mRNA was increased without any stimulation (Fig. 1B, ○◊∆) as shown in our previous studies (Mitsuhashi et al 2006). This result indicated that the values of time = 0 were not appropriate control, and vehicle control was used throughout except Figure 1A-C.…”

“…The Ct values of RNA34 were converted to copy number, and the % recovery was calculated in each well. For cellderived mRNA, the Ct values were converted to the copy number using respective calibration curves, and these values were further converted to copies/µl blood by dividing with % recovery of RNA34 in each sample (Mitsuhashi et al 2006). Each copy number of drug-treated triplicate samples was divided by the mean copy number of control samples (vehicle-treated ones) to calculate the mean ± standard deviation of the values of the fold increase.…”

“…The next challenge is to identify abnormal levels of mRNA that commonly exist in both normal and disease states. Although normal reference values of these mRNA were quantifi ed using healthy subject population (MAQC Consortium, 2006;Mitsuhashi et al 2006;Peters et al 2007;Zheng et al 2006), the identifi cation of disease-specifi c mRNA levels is one of the current topics in molecular diagnostics development.…”

mRNA of the pro-apoptotic gene BBC3 serves as a molecular marker for TNF-α-induced islet damage in humans

“…Differences in Cycle threshold (C t ) between the target and control mRNA (β actin) (ΔC t ) were used to quantify the relative amount of each target, calculated as 2 ÀΔC t . Primers used for gene expression assays have been previously described [25][26][27][28].…”

Posttransplantation Bone Marrow Assessment by Quantifying Hematopoietic Cell–Derived mRNAs in Plasma Exosomes/Microvesicles

“…Previously we introduced a unique system of leukocyte mRNA analysis from whole blood, in which leukocytes were trapped on a 96-well filter plate and leukocyte lysate was transferred to a 96-well oligo(dT)-immobilized microplate for direct poly(A) ϩ mRNA purification followed by cDNA synthesis and PCR (7)(8)(9). In this study, the leukocyte-capture filter plate was replaced with the newly developed EMV-capture filter plate, and HPC-derived mRNAs were quantified in plasma.…”