Evaluation of several methodological challenges in circulating miRNA qPCR studies in patients with head and neck cancer.
Author(s): Poel D, Buffart TE, Oosterling-Jansen J, Verheul HM, Voortman J
Publication: Exp Mol Med, 2018, Vol. 50, Page e454
PubMed ID: 29520111 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of processing delay, addition of carrier RNA during extraction, and normalization method on the measurement of circulating microRNA (ci-miRNA) levels in plasma and serum specimens.
Conclusion of Paper
Circulating miRNA levels in serum and plasma increased with the addition of glycogen or MS2 alone and in combination but the effect was generally lowest when both carriers were used in combination. Normalization of circulating miRNA levels to spiked-in cel-miR-39-3p and endogenous miR-16-5p resulted in lower variability in CT than normalization to either alone in both plasma and serum.
Processing delays resulted in increased hemolytic index in serum and a more than 1.5-fold increase in the levels of miR-21-5p in two of the five serum specimens and a decrease in miR-142-3p in two different serum specimens. In contrast, processing delay did not affect the hemolysis index of plasma specimens or levels of miR-21-5p and miR-92a-3p, but levels of miR-142-3p decreased by more than 1.5-fold in one of five specimens and increased more than 1.5-fold in three of five specimens, depending on time-point.
Intra-individual variability in ci-miRNA levels was small and limited to a few specimens.
Studies
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Study Purpose
The purpose of this study was to investigate the effects of adding glycogen or MS2 carrier RNA alone or in combination on the CT values for circulating miRNA in serum and plasma and to determine the best normalization strategy for ci-miRNA. Blood was collected from 10 head and neck squamous cell carcinoma (HNSCC) patients into plain BD Vacutainer tubes and BD Vacutainer EDTA tubes. Serum was obtained by centrifugation at 1500 x g for 10 min and plasma by centrifugation at 120 x g for 20 min followed by 360 x g for 20 min and 2700 x g for 10 min. Plasma and serum were pooled and stored at -80˚C. RNA was isolated in triplicate using the miRCURY RNA isolation kit with either no carrier, glycogen alone, MS2 alone, or a combination or glycogen and MS2. Cel-miR-39-3p was spiked-in during lysis. Extracted RNA was stored at -80˚C. Levels of miRNA were quantified by real-time PCR using the miRCURY LNA Universal RT microRNA kit. miRNA levels were normalized to cel-miR-39-3p or miR-16-5p alone or in combination.
Summary of Findings:
The mean CT values for circulating miRNAs in serum decreased with the addition of glycogen (1.11±0.56), MS2 (2.36±0.34), and glycogen with MS2 (1.53± 0.63) but the magnitude of the decrease was miRNA-dependent. Similarly, the mean CT of circulating miRNAs in plasma decreased with the addition of glycogen (1.17±0.43), MS2 (0.83±0.14), and glycogen with MS2 (1.11± 0.15). As the addition of MS-2 also reduced the standard error of the mean (SEM) of the average CT values for all of the miRNAs tested in serum and two of five miRNAs in plasma, it was chosen for all other experiments.
Normalization of circulating miRNA levels to spiked-in cel-miR-39-3p and endogenous miR-16-5p resulted in lower variability in CT than normalization to either alone in both plasma and serum.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Blood and blood products Plasma
Serum
Analyte Extraction and Purification Analyte isolation method Glycogen added
MS2 and Glycogen added
MS2 added
Real-time qRT-PCR Specific Data handling Normalized to cel-miR-39-3p and miR-16-5p
Normalized to cel-miR-39-3p
Normalized to miR-16-5p
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Study Purpose
This study investigated the effects of processing delay on the hemolysis index and levels of miRNA in serum and plasma specimens. Blood was collected from five healthy patients into four plain BD Vacutainer tubes and four BD Vacutainer EDTA tubes. To investigate the effects of delayed centrifugation, blood in plain tubes were stored on ice for 1, 2, 4, and 24 h before isolation of serum by centrifugation at 1500 x g for 10 min. EDTA blood was stored at room temperature for 1, 2, 4, and 24 h before isolation of plasma by centrifugation at 120 x g for 20 min followed by 360 x g for 20 min and 2700 x g for 10 min. One millileter aliquots of plasma and serum were stored at -80˚C. RNA was isolated using the miRCURY RNA isolation kit with the addition of DNAse. Cel-miR-39-3p was spiked-in during lysis. Extracted RNA was stored at -80˚C. Levels of miRNA were quantified by real-time PCR using the miRCURY LNA Universal RT microRNA kit. miRNA levels were normalized to a combination of cel-miR-39-3p and miR-16-5p and the threshold for differences was set to 1.5-fold. Hemolytic index was determined in plasma and serum specimens using the Roche Modular Cobas 8000 automated platform.
Summary of Findings:
Allowing blood to clot for 2 h or more rather than 1 h resulted in increased hemolysis (P=0.040) and storage for 24 h resulted in a more than 1.5-fold increase in the levels of miR-21-5p in two of the five serum specimens and a decrease in miR-142-3p in two other serum specimens. As expected, levels of cel-miR-39 in serum were unaffected by processing delay. The hemolysis index was weakly correlated with CT values for miR-16-5p (r2=0.2643, P=0.02) and miR-21-5p (r2=0.2661, P=0.02) in serum but only non-significantly correlated with miR-92a-3p (r2=0.1690, P=0.07).
All plasma specimens were verified to be platelet-free. Storage of blood for 24 h before centrifugation had no effect on the hemolysis index of plasma and CT for cel-miR-39-3p. Further, changes in levels of miR-21-5p and miR-92a-3p were less than 1.5-fold when centrifugation was delayed by up to 24 h. Levels of miR-142-3p decreased in one of five specimens when centrifugation was delayed by 2 h or 4 h and increased in three of five specimens when centrifugation was delayed by 24 h.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Protein Hematology/ auto analyzer RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Blood and blood products Plasma
Serum
Storage Storage duration 1 h
2 h
4 h
24 h
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
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Study Purpose
This study investigated intra-individual variability in ci-miRNA in HSNCC patients and healthy individuals. Blood specimens were obtained from 10 patients with HNSCC and five healthy individuals at two timepoints approximately 1 week apart. Serum was obtained by centrifugation at 1500 x g for 10 min and plasma by centrifugation at 120 x g for 20 min followed by 360 x g for 20 min and 2700 x g for 10 min. Plasma and serum were pooled and stored at -80˚C. RNA was isolated in triplicate using the miRCURY RNA isolation kit with MS2 and glycogen. Cel-miR-39-3p was spiked-in during lysis. Extracted RNA was stored at -80˚C. Levels of miRNA were quantified by real-time PCR using the miRCURY LNA Universal RT microRNA kit. miRNA levels were normalized to a cel-miR-39-3p and miR-16-5p in combination.
Summary of Findings:
Short term variation in all miRNAs was generally less than 1 cycle, but more than 1.5 cycles differences were noted for miR-21-5p levels in the plasma of a HNSCC and a healthy patient and for miR-92a-3p levels in the plasma of a HNSCC patient and the serum of another HNSCC patient.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Blood and blood products Plasma
Serum
Biospecimen Acquisition Time of biospecimen collection Collection 1
Collection 2
Preaquisition Diagnosis/ patient condition HNSCC
Normal