Droplet Digital PCR zur Quantifizierung von miR-21, miR-208a und miR-499 in der Diagnostik von Patienten mit funktionell relevanter koronarer Herzkrankheit

Abstract: Background
MicroRNAs (miRNAs) are small, non-coding RNA-molecules that are involved in post-translational regulation via alteration of messenger RNA (mRNA) and influencing of gene expression. In multiple studies, circulating miRNAs have shown potential as biomarkers of various diseases, including cardiovascular. Physiological and pathological changes in miRNA expression are traceable in serum samples. The stability of miRNAs in serum as well as changes in miRNA concentrations linked to pathology characterize miRNAs as interesting biomarkers in different diseases.
Recently, droplet digital polymerase chain reaction (ddPCR) has been established as a method of quantification. In ddPCR, the sample is divided in about 20.000 reactions. Using Poisson-statistics of positive to negative reactions, absolute quantification is possible, without using a standard curve. Compared to quantitative real time PCR (qRT-PCR), ddPCR shows a greater sensitivity as well as decreased day-to-day variability. Due to these superior technical qualities some problems that have previously occurred in quantifying miRNAs could be mitigated.
In previous studies, the oncomir miR-21, the cardiac-specific miR-208a and the cardiac-and skeletal muscle-specific miR-499 have shown to be alternated in cardiovascular disease. In this study we wanted to investigate concentration changes of these three miRNAs in patients with functional relevant coronary artery disease (CAD) as well as exercise-induced changes in microRNA concentration.
Methods and Results
In the first part of this study, multiple step dilution series of synthetic miRNA-21, miRNA-208a, miRNA-499 and C.elegans-miRNA-39 were used to assess the applicability of ddPCR to quantify miRNAs. All investigated miRNAs showed a great linearity (miR-21 r2=0.98; miR-208a r2=0.97; miR-499 r2=0.99; C.elegans-miR-39 r2=0.98). By calculating the limit of detection (LoD) and the limit of quantification (LoQ) with help of the dilution series we could show that even small amounts of miRNA are reliably quantified using ddPCR.
In the second part of this study we evaluated the use of miRNAs as diagnostic and prognostic biomarkers in functional relevant CAD. To do so, we included 200 consecutive patients in this study who had been admitted to the University Hospital Basel, Switzerland, in between July 2010 to June 2011 in order to assess the degree of functional relevant CAD. All patients were stressed, ergometrically, pharmacologically or combined ergometrically and pharmacologically; venous blood was drawn before stress, immediately after stress and 2h as well as 4h after stress. The functional relevance of CAD was estimated using myocardial-perfusion single-photon emission computed tomography (MPI-SPECT) or coronary angiography. hs-cTnI as a reference marker was determined in all patients using the Erenna-Assay. RNA was isolated from the serum samples of all patients, reverse transcribed and the final miRNA concentrations were quantified using ddPCR. All serum samples were spiked with C.elegans-miR-39 to normalize the samples.
We could show that neither miR-21, nor miR-208a or miR-499 differs significantly in patients with versus patients without functional relevant CAD. Furthermore, there was no significant difference in stress-induced changes in miRNA concentrations in patients with versus patients without functional relevant CAD. Moreover, there was no significant correlation of miR-21, miR-208a and/or miR-499 with all-cause mortality or the occurrence of a major cardiovascular event (MACE) such as myocardial infarction (MI) or sudden cardiac death. On the other hand, hs-cTnI concentrations were significantly higher in patients with functional relevant CAD versus patients without functional relevant CAD and showed even prognostic utility as higher hs-cTnI concentrations significantly correlated with all-cause mortality and MACE.
Conclusion
We were able to demonstrate that ddPCR is a reliable and applicable technique for the quantification of miRNAs where even small amounts of miRNA can be detected easily. In the second part of this study we were able to show that there is no utility of miR-21, miR-208a or miR-499 at rest, after stress or stress-induced changes for the diagnostics of CAD and that there is no prognostic value of these miRNAs regarding all-cause mortality of the occurrence of MACE

Object Details

• Location: Deutsche Nationalbibliothek Frankfurt am Main
• Extent: Online-Ressource
• Language: Deutsch
• Notes: Universität Freiburg, Dissertation, 2020

Classification and Topics

• Keyword: Koronare Herzkrankheit ; Herzkrankheit ; Patient ; Herzinfarkt ; SPECT ; Koronare Herzkrankheit ; miRNS

Contributors, Places and Time

• Event: Veröffentlichung
• (where): Freiburg
• (who): Universität
• (when): 2020
• Creator: Benning, Louise
• Contributor: Ahrens, Ingo ; Hortmann, Marcus Sebastian ; Albert-Ludwigs-Universität Freiburg. Medizinische Fakultät

Further information

• DOI: 10.6094/UNIFR/155316
• URN: urn:nbn:de:bsz:25-freidok-1553168
• Rights: Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Last update: 14.08.2025, 10:50 AM CEST