Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Plasma microRNA detection standardization test.

Author(s): Feng X, Liu Y, Wan N

Publication: J Clin Lab Anal, 2019, Vol. , Page e23058

PubMed ID: 31617231 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of extraction method used, plasma volume, timing of blood collection relative to eating, anticoagulant type, plasma storage temperature, and hemolysis on miRNA levels in plasma and the resultant myocardial risk score. The relative contributions of reverse-transcription (RT), PCR, and extraction timing on variability were also investigated. Unless otherwise noted below, venous blood was collected into K₂EDTA vacuum tubes and plasma was obtained by centrifugation at 2862 × g for 15 min at 4°C. Plasma was placed in RNAse-Free tubes and stored at -80°C until extraction using the EasyPure miRNA Kit. miRNA was reverse-transcribed using Transcript One‐Step gDNA Removal and cDNA Synthesis SuperMix Kit. Levels of miR‐126 and miR‐92a were quantified by real-time RT-PCR. The effects of extraction kit were evaluated by extracting miRNA from K₂EDTA plasma of three heathy individuals using the EasyPure miRNA Kit or Trizol LS reagent. The effects of plasma volume were investigated by using 100, 200, 300, 400, or 500 µL aliquots of three plasma specimens as input for the EasyPure miRNA Kit. To determine the effect of fasting, K₂EDTA blood was obtained from three healthy individuals 0, 1, 2, and 3 h after eating. The effects of anticoagulant were investigated by collection of blood from three healthy patients in sodium citrate, lithium heparin, and K₂EDTA tubes. To test the effects of storing plasma at different temperatures, plasma was stored for 1 week at -80°C and at 4°C. To test the effects of hemolysis, red blood cell lysate was diluted to obtain the appropriate levels and added to plasma. The effects of reverse-transcription, PCR, and extraction on the variance were tested by repeating processes for specimens. For extraction the variance between days was also compared with that within a day. The effects of reverse-transcription, PCR, and extraction on the variance was confirmed using specimens from 23 heathy individuals and 8 individuals with myocardial infarct.

   Summary of Findings:

   Significantly higher levels of miR-92a and miR-126 were found in all 3 specimens when RNA was extracted from plasma specimens using EasyPure rather than Trizol (P<0.05, all), but levels of U6 were not significantly different. The highest recovery of cel-39 miRNA occurred when plasma volume was 200-300 µL but the effects was only significant for 2 of the 3 specimens. Levels of miRNA were affected by the time relative to eating (P<0.05) with some miRNAs increasing and others decreasing, depending on timepoint, and thus affecting the risk score. While miRNA levels and risk scores were comparable for specimens collected in K₂EDTA and sodium citrate tubes, levels were lower in lithium heparin tubes (authors report Cq values >35). The authors report miRNA levels declined by 2.76 cycles when plasma was stored at 4°C rather than -80°C and the risk scores declined by approximately 5. Hemolysis resulted in a linear decrease in the Cq values for miR‐126, miR‐92a, and U6 but had no effect on the spiked-in control cel-miR-39 or on the risk score. Most of the variability in miRNA quantification was attributed to extraction steps (~0.5 cycles within a day and ~1 cycle between days), with differences of <0.2 cycles attributed to reverse-transcription and PCR steps. A similar trend of higher variability due to extraction than reverse-transcription or PCR was also observed when the study was repeated using specimens from healthy patients and those with myocardial infarction.

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Cardiovascular Disease
   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Healthy Myocardial infarction
   Biospecimen Acquisition	Anticoagulant	Lithium heparin Potassium EDTA Sodium citrate
   Biospecimen Acquisition	Time of biospecimen collection	Fasting 1 h after eating 2 h after eating 3 h after eating
   RT-PCR Specific	Targeted nucleic acid	miR‐126 miR‐92a U6 cel-miR-39
   Biospecimen Aliquots and Components	Aliquot size/volume	100 µL 200 µL 300 µL 400 µL 500 µL
   Storage	Storage temperature	4°C -80°C
   Analyte Extraction and Purification	Analyte isolation method	EasyPure miRNA Trizol LS reagent
   Biospecimen Aliquots and Components	Hemolysis	Hemolysate added

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