Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Optimization and Standardization of Circulating MicroRNA Detection for Clinical Application: The miR-Test Case.

Author(s): Marzi MJ, Montani F, Carletti RM, Dezi F, Dama E, Bonizzi G, Sandri MT, Rampinelli C, Bellomi M, Maisonneuve P, Spaggiari L, Veronesi G, Bianchi F, Di Fiore PP, Nicassio F

Publication: Clin Chem, 2016, Vol. 62, Page 743-54

PubMed ID: 27127244 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared real-time PCR quantified miRNA levels and inter-and intra-assay variability of both raw and normalized data collected from a range of serum input volumes that were extracted using one of two different miRNA purification columns.Blood was collected into S-Monovette tubes with clot activator from 972 healthy and lung cancer patients in the COSMOS lung cancer screening trial after a fast longer than 3 h. The first 3 mL of blood collected was discarded. Blood was allowed to clot for 3 h at room temperature before separation of plasma by centrifugation at 1000 g for 10 min. Serum was then transferred to cryotubes and snap-frozen on dry ice prior to storage at -80°C. To test the effects of purification method, RNA was extracted from two serum specimens using TriZol-LS and then purified with miRVANA columns or miRNeasy columns both manually and using a QIAcube. To test the effect of serum volume, RNA was purified from two 100, 200, 300, 400 and 500 µL serum aliquots using miRNeasy columns with the QIAcube instrument. For all other experiments, RNA was extracted from 300 µL serum using TriZol-LS and purified from 350 µL of the aqueous phase using the miRNeasy Mini Kit and a QIAcube machine. miRNAs were reverse transcribed using the MicroRNA Reverse Transcription Kit and pre-amplified using TaqMan PreAmp before real-time PCR amplification of miR-16, miR-19b and spiked-in miR-34a (positive control) or use of TaqMan low density arrays containing primers for 48 miRNAs of interest (6 housekeeping, 34 of interest and 8 controls). The control miRNA in the TaqMan assays included: three miRNAs with no or extremely low serum levels (miR-34a, miR-159 and cel-miR-39), three miRNAs expressed in serum known to be involved in other diseases (miR-21, miR-122, and miR-155), and two small RNAs (RNU44, RNU48).  The hemolysis index was measured by spectrophotometer.

   Summary of Findings:

   Cq values were lower and less variable when RNA was purified using miRNeasy columns manually or using QIAcube rather than miRVANA columns. Endogenous miRNA levels increased with increasing serum input up to 400 µL; however, a miRNeasy column input volume of 500 µL did not increase endogenous miRNA levels and resulted in lower recovery of spiked in miR-34 compared to volumes between 100-400 µL.  Collectively, data suggests that volumes >400 µL overload the column. Pre-amplification prior to real-time PCR lowered mean CT from 31 to 23 while remaining linear. A panel of 13 biomarkers and 6 housekeeping genes were found to have low Cq (<30, mean 21.9) and very strong correlations in serial dilution experiments (R²>0.90). The remaining miRNA were considered to have failed validation and were excluded from further analysis. Inter-assay variance for the whole assay was low (<0.2 Cq), with more variance attributed to extraction than real-time PCR or pre-amplification (P<0.01). Nevertheless, when extraction was performed on different days the variance remained low. Normalization of the raw data to either the 6 housekeeping genes or to the spike-in control reduced the variability in levels of the 13-diagnostic miRNA chosen for the miR-Test. The low inter-assay variability was confirmed by repeating the extraction step with 12 serum specimens from healthy individuals and with 12 serum specimens from patients with asymptomatic lung cancer. In specimens from 972 patients in the COSMOS study, the authors found a difference in extraction efficiency between the first extraction of the day and subsequent extractions which seemed to correspond with a shift in the machine temperature and humidity.  However, this effect was attenuated when real-time PCR miRNA levels were normalized to the 6 stable housekeeping genes that were identified.

   Biospecimens

   • Bodily Fluid - Serum

   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	Aliquot size/volume	100 µL, 200 µL, 300 µL, 400 µL, 500 µL,
   Real-time qRT-PCR Specific	Nucleic acid amplification	Pre-amplified Not pre-amplified
   Analyte Extraction and Purification	Analyte isolation method	miRNeasy manual miRNeasy automated on QIAcube miRVANA

   View more

2. Study Purpose

   This study investigated the effects of a patient’s fasting status, serum clot time (1, 2 or 3 h at room temperature), and serum storage temperature (overnight at 4°C or -80°C).  The effect of fasting was evaluated by collecting blood from 4 patients after an overnight fast and again 1, 2, and 3 h after eating. The effect of clot time was investigated by allowing case-matched samples from three healthy individuals to clot for 1, 3 or 6 h before serum separation. The effect of serum storage temperature was investigated by storing three aliquots of serum from two individuals at -80°C or 4°C overnight before analysis. Effects of hemolysis were investigated by adding different amounts of erythrocyte lysate to aliquots of a single serum specimen. For all experiments, including the 972 healthy patients and patients with lung cancer in the COSMOS cohort, the first 3 mL of blood was discarded and subsequent blood was collected into S-Monovette tubes with clot activator. Unless otherwise specified, patients were instructed to fast for >3 h prior to blood collection.  Blood was allowed to clot for 3 h at room temperature before serum was separated by centrifugation at 1000 g for 10 min. Serum was transferred to cryotubes and snap-frozen on dry ice prior to storage at -80°C, unless otherwise specified. RNA was extracted from 300 µL serum using TriZol-LS and purified from 350 µL of the aqueous phase using the miRNeasy Mini Kit and a QIAcube machine. miRNA were reverse transcribed using the MicroRNA Reverse Transcription Kit and pre-amplified using TaqMan PreAmp before real-time PCR amplification using TaqMan low density arrays containing primers for 48 miRNAs of interest (6 housekeeping, 34 of interest and 8 controls). The control miRNA in the TaqMan assays included: three miRNAs with no or extremely low serum levels (miR-34a, miR-159 and cel-miR-39), three miRNAs expressed in serum known to be involved in other diseases (miR-21, miR-122, and miR-155) and two small RNAs (RNU44, RNU48).  The hemolysis index was measured by spectrophotometer.

   Summary of Findings:

   Levels of the 13 biomarkers included in the miR-Test changed based on patient fasting status, as calculated lung cancer risk was lower when serum was collected 1 or 2 h after eating rather than after fasting.  In most samples, miR-Test scores were also lower 3 h after eating than after an overnight fast. Increasing the clot time from 1 to 3 or 6 h did not largely affect the levels of the 13 miRNA biomarkers in the miR-Test. However, storage of serum overnight at 4°C, rather than -80°C, had a much larger effect on the miRNA levels. Levels of the 13 miRNA biomarkers and 3 of the 6 housekeeping genes increased with increasing amounts of erythrocyte lysate, although the authors report that the calculated miR-Test score was not affected since the housekeeping genes also changed. The authors identified a hemolysis sensor based on the ratio of the expression of the three miRNAs that increased with hemolysis (miR-15b, miR-19a, and miR-19b) and a housekeeping gene that was unaffected by hemolysis (miR-146a); this hemolysis sensor was very strongly correlated to the degree of hemolysis that occurred in a sample (r=0.91). In the larger cohort of 972 specimens, when specimens were broken down by quartile for hemolysis sensor value, there was no apparent effect of hemolysis sensor score on the miR-Test score.

   Biospecimens

   • Bodily Fluid - Serum
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Protein	Spectrophotometry
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Time of biospecimen collection	Fasting 1 h after eating 2 h after eating 3 h after eating
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated
   Biospecimen Aliquots and Components	Hemolysis	Hemolysate added No hemolysate added
   Storage	Storage temperature	4°C -80°C
   Storage	Storage duration	1 h 3 h 6 h

   View more