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. 2016:2016:2901938.
doi: 10.1155/2016/2901938. Epub 2016 Sep 20.

Optimized Collection Protocol for Plasma MicroRNA Measurement in Patients with Cardiovascular Disease

Chi-Sheng Wu  1 , Fen-Chiung Lin  2 , Shu-Jen Chen  1 , Yung-Lung Chen  3 , Wen-Jung Chung  3 , Cheng-I Cheng  3
Affiliations

Optimized Collection Protocol for Plasma MicroRNA Measurement in Patients with Cardiovascular Disease

Chi-Sheng Wu et al. Biomed Res Int. 2016.

Abstract

Background. Various microRNAs (miRNAs) are used as markers of acute coronary syndrome, in which heparinization is considered mandatory therapy. Nevertheless, a standard method of handling plasma samples has not been proposed, and the effects of heparin treatment on miRNA detection are rarely discussed. Materials and Method. This study used quantitative polymerase chain reaction (qPCR) analysis to investigate how storage temperature, standby time, hemolysis, and heparin treatment affect miRNA measurement in plasma samples from 25 patients undergoing cardiac catheterization. Results. For most miRNAs, the qPCR results remained consistent during the first 2 hours. The miRNA signals did not significantly differ between samples stored at 4°C before processing and samples stored at room temperature (RT) before processing. miR-451a/miR-23a ratio < 60 indicated < 0.12% hemolysis with 100% sensitivity and 100% specificity. Pretreatment with 0.25 U heparinase I recovered qPCR signals that were reduced by in vivo heparinization. Conclusions. For miRNA measurement, blood samples stored at RT should be processed into plasma within 2 hours after withdrawal and should be pretreated with 0.25 U heparinase I to overcome heparin-attenuated miRNA signals. The miR-451a/miR-23a ratio is a reliable indicator of significant hemolysis.

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Conflict of interest statement

No author has any commercial associations or interests in this research, including consultancies, stock ownership, or other competing equity interests or patent-licensing arrangements.

Figures

Figure 1
Figure 1
Flowchart of plasma sample collection procedure. Plasma samples were collected at the catheterization laboratory as described in Materials and Methods.
Figure 2
Figure 2
Expressions of miR-425a and miR-23a represent the hemolysis status of clinical samples. The qPCR analysis revealed that expressions of miR-451a and miR-23a stored at (a) RT or (b) at 4°C were stable from 0 h to 8 h. The Ct values shown in the tables indicate the qPCR results for five individuals at different time points. The P value indicates the significance of each time point compared with time 0 h. (c) Manual hemolysis test was performed in 32 differential hemolytic plasma samples (see Materials and Methods). Hemolytic grade was defined by hemolysis card (Supplementary Fig.  1A). The miR-425a/miR-23a ratio increased as hemolysis grade increased.
Figure 3
Figure 3
Plasma storage condition test. The miRNA expressin patterns at time 0 h, 0,5 h, 1 h, 2 h, and 4 h for storage at (a) RT and (b) at 4°C. (c) Average change in Ct value in five clinical samples of plasma stored for varying durations ranging from 0.5 h to 4 h at RT and at 4°C. From 0.5 h to 2 h, the delta Ct was more stable at RT than at 4°C. The scale indicates the delta Ct value of the qPCR result in comparison with time 0.
Figure 4
Figure 4
Comparison of samples stored at RT for 0 h and at RT for 2 h. The qPCR results at time 0 and at 2 h were compared in nine individual plasma samples.
Figure 5
Figure 5
Heparinase I treatment improved Ct values of qPCR signals of clinical samples. (a) The qPCR signal was enhanced by treatment with 0.5 U and 0.25 U of heparinase I in spike-in control cel-miR-39 in four individual plasma samples stored for 0.5 h (top) or at 8 h (bottom) at RT or at 4°C. (b) Two individual plasma samples (P03 ad P04) treated with 0.5 U and 0.25 U heparinase I showed similar qPCR detection results for four independent miRNA targets. (c) Heparinase I treatment improved the qPCR signal. For cel-miR-39 (top), a dose-dependent effect of heparinase I was obsercved in samples stored for 2 h or 4 h at RT or at 4°C. Similar results were observed in four individual samples of human miR-15b-5p. (d) A heparinase I dose of 0.25 U significantly improved the qPCR signal in samples stored for 2 h or 4 h at RT or at 4°C in one-way ANOVA. ∗∗∗ P < 0.001; ∗∗ P < 0.01; ∗ P < 0.05.
Figure 6
Figure 6
Conservative correlation of miRNA expression induced by heparinase I treatment. (a) In four individuals, treatment with 0.25 U heparinase I induced similar miRNA expression levels in samples stored for 2 h at RT or at 4°C (top). Similar results were observed in samples stored for 4 h (bottom). (b) One-way ANOVA showed that miRNA expression induced by 0.25 U heparinase I treatment significantly correlated with storage time and storage temperature.
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References

    1. Finegold J. A., Asaria P., Francis D. P. Mortality from ischaemic heart disease by country, region, and age: statistics from World Health Organisation and United Nations. International Journal of Cardiology. 2013;168(2):934–945. doi: 10.1016/j.ijcard.2012.10.046. - DOI - PMC - PubMed
    1. Jousilahti P., Vartiainen E., Tuomilehto J., Puska P. Sex, age, cardiovascular risk factors, and coronary heart disease: a prospective follow-up study of 14 786 middle-aged men and women in Finland. Circulation. 1999;99(9):1165–1172. - PubMed
    1. Gueyffier F., Boissel J. P., Pocock S., et al. Identification of risk factors in hypertensive patients: contribution of randomized controlled trials through an individual patient database. Circulation. 1999;100(18):e88–e94. - PubMed
    1. Jani B., Rajkumar C. Ageing and vascular ageing. Postgraduate Medical Journal. 2006;82(968):357–362. doi: 10.1136/pgmj.2005.036053. - DOI - PMC - PubMed
    1. Hawkins R. Discrepancy between visual and spectrophotometric assessment of sample haemolysis. Annals of Clinical Biochemistry. 2002;39(5):521–522. doi: 10.1258/000456302320314575. - DOI - PubMed

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