Establishment of an easy and straight forward heparinase protocol to analyse circulating and myocardial tissue micro-RNA during coronary artery-bypass-graft surgery

Engler, Andrea; Dreja, Florian; S, Köberle; Thielmann, Matthias; Frey, Ulrich H.

doi:10.1038/s41598-018-19748-6

Cited by 10 publications

(9 citation statements)

References 44 publications

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“…Only the RNA samples with 260/280 ratios of 1.8 to 2.0 were used in the study. The isolated plasma miRNA (20 µl vol) was incubated with 1 U (1 µl vol) of heparinase I (St. Louis, MO) [ 7 ] and 20 U (1 µl vol) RiboLock RNase inhibitor (Thermo Fisher Scientific) at room temperature for 30 minutes. This is a critical step to minimize the inhibitory heparin from all the patient-derived plasma.…”

Section: Methodsmentioning

confidence: 99%

Cellular miR-150-5p may have a crucial role to play in the biology of SARS-CoV-2 infection by regulating nsp10 gene

2021

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The role for circulating miRNAs as biomarkers of the COVID-19 disease remains uncertain. We analysed the circulating miRNA profile in twelve COVID-19 patients with moderate-severe disease. This analysis was conducted by performing next generation sequencing (NGS) followed by real-time polymerase chain reaction (RT-qPCR). Compared with healthy controls, we detected significant changes in the circulating miRNA profile of COVID-19 patients. The miRNAs that were significantly altered in all the COVID-19 patients were miR-150-5p, miR-375, miR-122-5p, miR-494-3p, miR-3197, miR-4690-5p, miR-1915-3p, and miR-3652. Infection assays performed using miRNA mimics in HEK-293 T cells determined miR-150-5p to have a crucial role in SARS-CoV-2 infection and this was based on the following data: (i) miR-150-5p mimic lowered in vitro SARS-CoV-2 infection; (ii) miR-150-5p inhibitor reversed the effects of miR-150-5p mimic on SARS-CoV-2 infection of cells; and (iii) a novel miRNA recognition element (MRE) was identified in the coding strand of SARS-CoV-2 nsp10 , the expression of which could be inhibited by miR-150-5p mimic. Our findings identified crucial miRNA footprints in COVID-19 patients with moderate-severe disease. A combination of co-transfection and Western blotting experiments also determined the ability of miR-150-5p to inhibit SARS-CoV-2 infection via directly interacting with MRE in the coding strand of nsp10 . Our investigation showed that a sharp decline in the miR-150-5p plasma levels in COVID-19 patients may support enhanced SARS-CoV-2 infection. Furthermore, this study provides insight into one possible mechanism by which COVID-19-induced changes to miR-150-5p levels may promote SARS-CoV-2 infection via modulating nsp10 expression.

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Section: Methodsmentioning

confidence: 99%

Cellular miR-150-5p may have a crucial role to play in the biology of SARS-CoV-2 infection by regulating nsp10 gene

2021

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“…Nevertheless, there are only few studies that have investigated the changes of miRNA profiles during cardiac surgery. Accordingly, the elevation of miR-1, mir-133 and miR-499 levels in plasma and atrial tissue are associated with myocardial damage in open-heart surgery with CPB [18][19][20]. Additionally, aberrant miRNA expression may implicate cardiac dysfunction, and thus, make them potential biomarkers and therapeutic targets during elective cardiac surgery.…”

Section: Discussionmentioning

confidence: 99%

MicroRNA Expression Profile Changes after Cardiopulmonary Bypass and Ischemia/Reperfusion-Injury in a Porcine Model of Cardioplegic Arrest

et al. 2020

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Identification of microRNAs (miRNA) associated with cardiopulmonary bypass, cardiac arrest and subsequent myocardial ischemia/reperfusion may unravel novel therapeutic targets and biomarkers. The primary aim of the present study was to investigate the effects of cardiopulmonary bypass and temperature of cardioplegic arrest on myocardial miRNA profile in pigs’ left ventricular tissue. We employed next-generation sequencing to analyse miRNA profiles in the following groups: (1) hearts were arrested with antegrade warm St Thomas Hospital No. 2 (STH2) cardioplegia (n = 5; STH2-warm, 37 °C) and (2) cold STH2 (n = 6; STH2-cold, 4 °C) cardioplegia. Sixty min of ischemia was followed by 60 min of on-pump reperfusion with an additional 90 min of off-pump reperfusion. In addition, two groups without cardiac arrest (off-pump and on-pump group; n = 3, respectively) served as additional controls. STH2-warm and STH2-cold cardioplegia revealed no hemodynamic differences. In contrast, coronary venous creatine kinase-myocardial band (CK-MB) levels were significantly lower in pigs receiving STH2-warm cardioplegia (p < 0.05). Principal component analysis revealed that cardiopulmonary bypass and cardioplegic arrest markedly affected miRNAs in left ventricular tissue. Accordingly, ssc-miR-122, ssc-miR-10a-5p, ssc-miR-193a-3p, ssc-miR-499-3p, ssc-miR-374a-5p, ssc-miR-345-5p, ssc-miR-142-3p, ssc-miR-424-5p, ssc-miR-545-3p, ssc-miR-30b-5p, ssc-miR-145-5p, ssc-miR-374b-5p and ssc-miR-139-3p were differently regulated by cardiopulmonary bypass (false discovery rate (FDR) < 0.05 versus off-pump group). However, only ssc-miR-451 was differently expressed between STH2-warm and STH2-cold (FDR < 0.05). These data demonstrate for the first time that cardiopulmonary bypass and temperature of cardioplegic solution affected the expression of miRNAs in left ventricular tissue. In conclusion, specific miRNAs are potential therapeutic targets for limiting ischemia-reperfusion injury in patients undergoing cardiac surgery.

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“…Plasma should be stored immediately in a freezer at −80°C, with miRNA content determined as soon as possible after extraction to minimize miRNA degradation. When collecting blood samples, use of lithium heparin anticoagulation tubes should be avoided, because heparin inhibits the activity of Taq DNA polymerase to affect the quantification of miRNA . Consequently, it is recommended to strictly follow the operating procedures when collecting and preparing samples.…”

Section: Discussionmentioning

confidence: 99%

Plasma microRNA detection standardization test

Feng

Liu

Wan

2019

Clinical Laboratory Analysis

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Background MicroRNAs (miRNAs) can be used for early diagnosis of myocardial infarction. However, due to a lack of standardized operating procedures, their value for clinical application is low. Methods Detection of plasma miRNAs was optimized by analyzing factors influencing miRNA variance and myocardial infarction risk scores during analysis (extraction, reverse transcription, and real‐time PCR) and pre‐analysis (dietary status, anticoagulants, storage conditions, and hemolysis). Results Regarding variable factors during analysis, the centrifugal column method was superior to Trizol LS reagent when extracting miRNA from plasma. Recovery rate was highest with plasma volumes of 200 and 300 µL. During analysis, the main source of miRNA detection inaccuracy was derived from RNA extraction (mainly organic extraction), and not reverse transcription or PCR. MiRNA variance could be reduced by use of an internal reference. During analysis, 95% of risk score variation fluctuated within a range of 6.267. The variable factors pre‐analysis mainly involved dietary status, anticoagulant selection, and storage conditions. Hemolysis positively correlated with miRNA levels, but there was no significant change in risk score after internal reference calibration. Conclusion Preliminary standardization for miRNA detection provides a reference for clinical blood testing of miRNAs.

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Establishment of an easy and straight forward heparinase protocol to analyse circulating and myocardial tissue micro-RNA during coronary artery-bypass-graft surgery

Cited by 10 publications

References 44 publications

Cellular miR-150-5p may have a crucial role to play in the biology of SARS-CoV-2 infection by regulating nsp10 gene

Cellular miR-150-5p may have a crucial role to play in the biology of SARS-CoV-2 infection by regulating nsp10 gene

MicroRNA Expression Profile Changes after Cardiopulmonary Bypass and Ischemia/Reperfusion-Injury in a Porcine Model of Cardioplegic Arrest

Plasma microRNA detection standardization test

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