It has long been thought that mRNA is labile and easily prone to degradation. However, a recent study demonstrated that GAPDH mRNA in cell-free plasma may remain stable up to 24 hours after blood collection. As there are no other independent studies, we attempted to reproduce the findings of that study. In our study, blood was collected from a healthy male volunteer into Vacutainer tubes containing EDTA. Blood samples were placed on ice and plasma separated by double-centrifugation at times 0, 1, 2, and 5 hours after blood collection. mRNA was extracted from four aliquots of the blood sample by means of the QIAamp Viral RNA kit. Extracted mRNA was converted to cDNA by reverse transcription before real time quantitative PCR measurement of the housekeeping beta-actin gene. Plasma beta-actin mRNA at 2 hours (0.012; 0.0031-0.0297, median and range) was significantly lower (P= 0.022) than at 0 hours (0.12; 0.057-0.165) (P= 0.016). The levels decreased further at 5 hours (0.0037; 0.0024-0.011) (P= 0.004). The results show that plasma beta-actin mRNA levels decrease with time after blood collection and that this is likely to be due to degradation in vitro.

Nucleic acids, both DNA and mRNA, have been detected in the circulation and have been demonstrated to be useful in such areas as fetal medicine, oncology, and transplantation. When mRNA is measured in circulating blood, the results are expressed in relation to a reference gene product in order to correct for any differences in extraction, volume of starting material or other differences. Many authors use β-actin mRNA and express results as a ratio of target mRNA to β-actin mRNA. We have used a similar approach when studying diabetic retinopathy. Recently, we planned to investigate the expression of thyroid dependent gene expression in acutely ill patients. As a control study, we examined the expression of thyroid hormone-dependent gene expression in subjects with hyperthyroidism and found that the expression of β-actin mRNA was affected by thyroid hormone status. Blood samples were taken into PAX gene TM tubes from 31 healthy subjects (mean age, 43 ± 16yrs) and 7 patients with hyperthyroidism (mean age, 43 ± 5yrs). Diagnosis of hyperthyroidism was confirmed by clinical findings and biochemical results. After extraction of mRNA, cDNA was synthesized using reverse transcription. Quantification of Na/K-ATPase, T3 receptor, and β-actin cDNA was carried out by RT-PCR. Median β-actin levels were significantly higher in hyperthyroid subjects compared to healthy subjects (18.2 versus 2.30; P < 0.00042). When mRNA for the T3 receptor was expressed in relation to β-actin, there was a significantly higher in hyperthyroidism (0.0168 versus 0.218, P < 0.05). However, this was significantly lower when expressed in relation to total RNA (12.2 versus 2.24, P < 0.00015).We conclude that normalizing results to β-actin may not be appropriate in all circumstances.