Quantification of Circulating miRNAs in Plasma: Effect of Preanalytical and Analytical Parameters on Their Isolation and Stability.

Author(s): Sourvinou IS, Markou A, Lianidou ES

Publication: J Mol Diagn, 2013, Vol. 15, Page 827-34

PubMed ID: 23988620 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of plasma volume, RNA precipitation temperature during TriZol LS extraction, and miRNA extraction kit type on the recovery of spiked cel-miR-39 and endogenous hsa-miR-21 in EDTA plasma from healthy individuals and patients with non-small cell lung carcinomas. EDTA blood was centrifuged and the was plasma spiked with cel-miR-39 before miRNA extraction.

   Summary of Findings:

   Using the TriZol LS kit, the highest recovery of cel-miR-39 and hsa-miR-21 from plasma was achieved when the least volume of plasma was used (200 uL) and when RNA was precipitated at room temperature for 10 min rather than at -20 degrees C overnight. The yields of cel-miR-39 and hsa-miR-21 were highest using the mirVana PARIS kit for extraction and lowest using TRIzol LS (with optimized protocol) or the Norgen miRNA purification kit.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	TRIzol LS miRNeasy mini kit mirVana PARIS kit Norgen miRNA purification kit
   Analyte Extraction and Purification	Analyte purification	Precipitation at room temperature Precipitation at -20 degrees C
   Biospecimen Aliquots and Components	Aliquot size/volume	200 uL 250 uL 300 uL 500 uL
   Real-time qRT-PCR Specific	Targeted nucleic acid	hsa-miR-21 cel-miR-39

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2. Study Purpose

   The purpose of this study was to determine the effects of storage temperature and duration for plasma or extracted miRNA on levels of cel-miR-39, hsa-miR-21 and hsa-miR-16 in EDTA plasma from healthy individuals and patients with non-small cell lung carcinomas. EDTA blood was centrifuged and plasma was spiked with cel-miR-39 before miRNA extraction. Plasma was stored for up to 4 months, while extracted cel-miR-39 was stored for up to 1 year.

   Summary of Findings:

   CT values for hsa-miR-21 and hsa-miR-16 increased with storage of plasma at 4, -20 or -70 degrees C, and significant increases, compared to 24 h of storage, were observed after 1 and 4 months at all three storage temperatures (p<0.05, all). However, the CT values of hsa-miR-21 and hsa-miR-16 increased by 3-4 cycles after storage of plasma at -20 or -70 degrees C, while the CT values of hsa-miR-21 and hsa-miR-16 increased by 14-15 cycles after storage of plasma at 4 degrees C for 4 months. Consequently, the authors conclude storage of plasma at -20 or -70 degrees C is better than storage of plasma at 4 degrees C. After extraction, cel-miR-39 was stable at -70 degrees C for at least 1 year.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Neoplastic - Carcinoma
   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	24 h 48 h 1 month 2 months 4 months 6 months 8 months 9 months 12 months
   Storage	Storage temperature	-70 degrees C -20 degrees C 4 degrees C
   Real-time qRT-PCR Specific	Targeted nucleic acid	hsa-miR-21 hsa-miR-16

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