Plasma components affect accuracy of circulating cancer-related microRNA quantitation.
Author(s): Kim DJ, Linnstaedt S, Palma J, Park JC, Ntrivalas E, Kwak-Kim JY, Gilman-Sachs A, Beaman K, Hastings ML, Martin JN, Duelli DM
Publication: J Mol Diagn, 2012, Vol. 14, Page 71-80
PubMed ID: 22154918 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to determine the effects of anticoagulant use and type, miRNA extraction and purification method, and analytical variables including template amount and polymerase type on the quantitation of miR-16 and miR-223 in plasma and serum. Diluted plasma and serum specimens were analyzed immediately or were flash-frozen and stored at -80 degrees C. After RNA extraction, samples were used immediately or stored at -70 degrees C. The stability of miR-16 (isolated from cultured BC3 cells) was evaluated during incubation of whole blood and plasma at 10 degrees C.
Summary of Findings:
The highest levels of miR-16 were quantified in NaF/KOx plasma followed by citrated plasma. Both of these specimen types yielded significantly higher levels of miR-16 than EDTA plasma or serum. The highest average levels of miR-223 were also found in NaF/KOx plasma, although the levels measured in serum specimens were highly variable, and in some cases, were higher than levels measured in NaF/KOx plasma. The addition of NaF and KOx to serum or to EDTA plasma tripled and doubled the quantified levels of miR-16, respectively (p=0.014 and p=0.037, respectively). PCR amplification efficiency of miR-16 or a synthetic RNA added during RNA extraction (SYNTH) was not significantly affected by the different anticoagulants used during plasma collection or in serum. miR-16 isolated from cultured BC3 cells was stable in EDTA whole blood at 10 degrees C for 2 h but not 17 h, but it was not stable in EDTA plasma for even 2 h. miRNA detection in EDTA plasma and serum was highest when miRNA was extracted by phenol/chloroform followed by silica membrane adsorption of RNA, but either of these treatments by themselves did not increase quantitation, and neither did the addition of detergents or ribonuclease inhibitors. The addition of heparinase to heparinated plasma significantly improved the detection of miR-16 by RT-PCR. Extraction of miRNA from 50 uL of EDTA plasma or serum improved miR-16 detection compared to when 10 or 200 uL were used. Extraction of miRNA from 10 uL of heparinated plasma yielded superior miR-16 detection than when 50 or 200 uL were used. Importantly, the use of Hemo KlenTaq during RT-PCR improved amplification of miR-16, but nonspecific cDNA amplification had to be overcome by the addition of intact Taq polymerases along with the Hemo KlenTaq.
Biospecimens
Preservative Types
- None (Fresh)
- Frozen
Diagnoses:
- AIDS/HIV-related
- Normal
- Neoplastic - Sarcoma
- Neoplastic
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR RNA RT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation None (fresh)
Snap frozen
Biospecimen Aliquots and Components Blood and blood products Plasma
Serum
Whole blood
Biospecimen Acquisition Anticoagulant EDTA
None
Sodium citrate
Sodium fluoride/potassium oxalate
Sodium heparin
Multiple concentrations evaluated
Real-time qRT-PCR Specific Template/input amount 10 uL
50 uL
200 uL
Real-time qRT-PCR Specific Nucleic acid amplification Phusion
Phire
Hemo KlenTaq
Standard Taq
TaqMan
Storage Storage duration 2 h
17 h
Analyte Extraction and Purification Analyte purification TRIzol only
TRIzol followed by one phenol/chloroform extraction
TRIzol followed by three phenol/chloroform extractions
Silica membrane adsorption
No silica membrane adsorption
Addition of detergents
Addition of heparinase I
No heparinase I
Analyte Extraction and Purification Nucleic acid digestion Addition of ribonuclease inhibitors
None