Stabilization of circulating thyroglobulin mRNA transcripts in patients treated for differentiated thyroid carcinoma

Abstract

Background The clinical utility of serum thyroglobulin in the follow-up of patients with differentiated thyroid carcinoma may be compromised by the presence of endogenous antithyroglobulin antibodies. To prevent interference by antithyroglobulin antibodies several groups have developed real-time PCR-based assays for quantification of blood thyroglobulin mRNA levels. For accurate quantification of thyroglobulin mRNA in blood preanalytical factors must be recognized and controlled. In this study, we evaluate the effect of different blood RNA stabilizing systems - the Tempus Blood RNA system and the PAXgene Blood RNA system - and storage time on RNA yield and quality, and thyroglobulin mRNA stability. Methods Blood samples from 11 patients previously treated for differentiated thyroid carcinoma were collected in K-EDTA, Tempus and PAXgene tubes and maintained at room temperature. RNA was isolated following storage for 0 and 72 h, and RNA yield, integrity and purity was determined. Thyroglobulin, GAPDH and ACTB mRNA levels were quantified by semi-quantitative real-time PCR. Results The RNA yield was significantly higher for blood collected in Tempus tubes compared with PAXgene tubes following storage for 72 h at room temperature ( P = 0.0011). High-quality RNA could be extracted from blood collected in PAXgene and Tempus tubes. Blood collected in K-EDTA tubes, but not in PAXgene and Tempus tubes, showed significant changes in thyroglobulin mRNA levels following storage for 72 h at room temperature ( P = 0.0263). Conclusions Stabilization of blood in PAXgene and Tempus tubes enables storage at room temperature for up to 72 h, without compromising thyroglobulin mRNA levels.

The Effect of Tropical Temperatures on the Quality of RNA Extracted from Stabilized Whole-Blood Samples

“…However, no significant differences in RIN at 25 • C (p = 0.787) or 40 • C (p = 0.399) was found for tube types (Figure 1A). In agreement with previously published studies [9,16], these results demonstrated that RNA integrity is temperature-sensitive, and that both tube types produced low-quality RNA at increased storage times and temperatures. Nevertheless, our data suggest that Tempus ™ tubes may provide better RNA integrity (higher RIN values) under certain suboptimal tropical conditions compared to PAXgene ® tubes.…”

“…This ratio is decreased in the presence of residual phenol, salts, and carbohydrates that can affect the accuracy of downstream application and used as a secondary measurement for RNA purity [38]. Historically, low A260/A230 ratios are reportedly attributed to the high salt content of the elution buffers contained in PAXgene ® extraction kits [16,17,34,35] and as well as in Tempus ™ extraction kits [16,22,39].…”

“…Commercially available blood collection systems with additives that stabilize RNA have been developed to address RNA stability, resulting in significantly enhanced quantity and quality of RNA extracted from whole blood [15,16]. The two most widely used blood RNA stabilizing systems are PAXgene ® and Tempus ™ [17,18].…”

Evaluation of RNA purification methods by using different blood stabilization tubes: identification of key features for epidemiological studies

“…With respect to the purity of the RNA, the best results were obtained with Methodology C. Overall, 'very good' and comparable purity values were observed for Methodology A and Methodology C, in accordance with previous research [9,12,20]. However, we observed more unstable A 260 /A 230 ratios with Methodology A and especially for Methodology B than these previous studies.…”

Detecting ALK, ROS1, and RET fusions and the METΔex14 splicing variant in liquid biopsies of non‐small‐cell lung cancer patients using RNA‐based techniques

“…The cfRNA was immediately isolated from 4 mL of the supernatant of the second centrifugation using the QIAsymphony ® DSP Virus/Pathogen Midi Kit in a QIAsymphony robot (Qiagen, Hilden, Germany), following the manufacturer's instructions. The maximum 24‐h interval between blood collection and RNA purification was decided based on a stability study performed in‐house (Table S3 ), which was in line with previous publications about RNA stability in K2‐EDTA tubes [ 22 , 23 , 24 , 25 ]. The automatic extraction procedure is based on magnetic particles and involves bind, wash, and elution steps.…”