Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Challenges in circulating miRNA analysis in adrenocortical tumors.

Author(s): Vékony B, Nyirő G, Butz H, Szeredás BK, Tóth V, Ferdinandy P, Patócs A, Igaz P

Publication: Endocr Relat Cancer, 2025, Vol. 32, Page

PubMed ID: 40472370 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the quantified levels of four endogenous microRNAs (miRNA, miR) and one spike-in miRNA using real-time PCR and ddPCR in matched K₂EDTA and K₃EDTA plasma. Blood was collected from 13 healthy volunteers and seven patients with endocrine disorders (diagnosis included primary aldosteronism, idiopathic hypoparathyroidism, nonfunctional pituitary adenoma, and hypothyroidism) into matched K₂EDTA Vacutainer and K₃EDTA Vacuette tubes. Blood was immediately placed on ice, and plasma was obtained by centrifugation at 1,861 g for 10 min at 4°C. miRNA was isolated from plasma using the miRNeasy Serum/Plasma Kit with the inclusion of cel-miR-39 as a spike-in. Extracted miRNA and plasma were stored at -80°C. RNA was quantified by spectrophotometer. Levels of cel-mir-39-3p, miR-16-5p, miR-21-5p, miR-210-3p and miR-483-5p were quantified using TaqMan real-time PCR assays and by ddPCR using the same TaqMan probes.

   Summary of Findings:

   Using either K₂EDTA or K₃EDTA plasma, real-time PCR cycle threshold (CT) values were correlated with ddPCR copy numbers for cel-miR-39-3p (K₂EDTA: R²=0.871982, P=8.72e^-11 and K₃EDTA: R²=0.433754, P=0.0016, respectively), miR-16-5p (R²=0.96138, P=8.55e^-15 and R²=0.96138, P=1.22e^-11, respectively), miR-21-5p (R²=0.877594, P=7.08e^-12 and R²=0.900411, P=3.05e^-13, respectively) and miR-210-3p (R²=0.37271, P=0.0042 and R²=0.917572, P=5.02e^-11, respectively), but no correlation was observed for miR-483-5p. Interestingly, while real-time PCR CT values that were normalized to cel-miR-39p were correlated with the ddPCR copy numbers for each of the four miRNAs when K₂EDTA plasma was used (R²=0.2786-0.3691, P<0.05, all), the correlation between normalized real-time PCR CT value and ddPCR copy number was limited to miR-210-3p when K₃EDTA plasma was used (R²=0.2359, P=0.0299). Bland-Altman plots revealed that, generally, real-time PCR measured higher levels than ddPCR at the lower end of the quantification range, but ddPCR measured higher levels than real-time PCR at the higher end of the range. Further, the bias associated with the quantification method was generally lower in K₂EDTA than K₃EDTA plasma and when CT values were normalized to cel-mir-39-3p.  There was much more variability in raw CT values and copy numbers in K₃EDTA plasma than K₂EDTA plasma. Additionally, K₃EDTA plasma had higher cel-miR-39-sp normalized (but not raw) levels of miR-16-5p (P=0.008219), and miR-210-3p (P=0.009439), higher raw CT (but not normalized) values for miR-483-5p (P=0.02965) and lower cel-miR-39-3p normalized (but not raw) levels of miR-21-5p (P=0.006142) than K₂EDTA plasma. Interestingly, ddPCR levels of each of the five miRNAs were found to be comparable between K₂EDTA and K₃EDTA plasma.

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Other diagnoses
   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Digital PCR
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Real-time qRT-PCR Specific	Data handling	Raw CT Normalized to cel-miR-39-3p
   Real-time qRT-PCR Specific	Technology platform	TaqMan real-time PCR ddPCR
   Biospecimen Acquisition	Anticoagulant	Multiple forms evaluated Potassium EDTA

   View more