Quantitative PCR Measurement of miR-371a-3p and miR-372-p Is Influenced by Hemolysis
Cell-free microRNAs have been reported as biomarkers for several diseases. For testicular germ cell tumors (GCT), circulating microRNAs 371a-3p and 372-3p in serum and plasma have been proposed as biomarkers for diagnostic and disease monitoring purposes. The most widely used method for quantificati...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2019-05-01
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| Series: | Frontiers in Genetics |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fgene.2019.00463/full |
| _version_ | 1818107574046687232 |
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| author | Mette Pernille Myklebust Benedikte Rosenlund Peder Gjengstø Bogdan Stefan Bercea Ása Karlsdottir Marianne Brydøy Olav Dahl Olav Dahl |
| author_facet | Mette Pernille Myklebust Benedikte Rosenlund Peder Gjengstø Bogdan Stefan Bercea Ása Karlsdottir Marianne Brydøy Olav Dahl Olav Dahl |
| author_sort | Mette Pernille Myklebust |
| collection | DOAJ |
| description | Cell-free microRNAs have been reported as biomarkers for several diseases. For testicular germ cell tumors (GCT), circulating microRNAs 371a-3p and 372-3p in serum and plasma have been proposed as biomarkers for diagnostic and disease monitoring purposes. The most widely used method for quantification of specific microRNAs in serum and plasma is reverse transcriptase real-time quantitative PCR (RT-qPCR) by the comparative Ct-method. In this method one or several reference genes or reference microRNAs are needed in order to normalize and calculate the relative microRNA levels across samples. One of the pitfalls in analysis of microRNAs from serum and plasma is the release of microRNAs from blood cells during hemolysis. This is an important issue because varying degrees of hemolysis are not uncommon in routine blood sampling. Thus, hemolysis must be taken into consideration when working with circulating microRNAs from blood. miR-93-5p, miR-30b-5p, and miR-20a-5p have been reported as reference microRNA in analysis of the miR-371a-373 cluster. We here show how these three microRNAs are influenced by hemolysis. We also propose a new reference microRNA, miR-191-5p, which is relatively stable in serum samples with mild hemolysis. In addition, we show how hemolysis can have effect on the reported microRNA levels in patient samples when these reference microRNAs are used in samples with varying levels of hemolysis. |
| first_indexed | 2024-12-11T02:01:37Z |
| format | Article |
| id | doaj.art-24b0b524b4ec476c829e195d5d10aa44 |
| institution | Directory Open Access Journal |
| issn | 1664-8021 |
| language | English |
| last_indexed | 2024-12-11T02:01:37Z |
| publishDate | 2019-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Genetics |
| spelling | doaj.art-24b0b524b4ec476c829e195d5d10aa442022-12-22T01:24:28ZengFrontiers Media S.A.Frontiers in Genetics1664-80212019-05-011010.3389/fgene.2019.00463453389Quantitative PCR Measurement of miR-371a-3p and miR-372-p Is Influenced by HemolysisMette Pernille Myklebust0Benedikte Rosenlund1Peder Gjengstø2Bogdan Stefan Bercea3Ása Karlsdottir4Marianne Brydøy5Olav Dahl6Olav Dahl7Department of Oncology and Medical Physics, Haukeland University Hospital, Bergen, NorwayDepartment of Oncology and Medical Physics, Haukeland University Hospital, Bergen, NorwayDepartment of Urology, Haukeland University Hospital, Bergen, NorwayDepartment of Urology, Helse Fonna, Haugesund, NorwayDepartment of Oncology and Medical Physics, Haukeland University Hospital, Bergen, NorwayDepartment of Oncology and Medical Physics, Haukeland University Hospital, Bergen, NorwayDepartment of Oncology and Medical Physics, Haukeland University Hospital, Bergen, NorwayDepartment of Clinical Science, University of Bergen, Bergen, NorwayCell-free microRNAs have been reported as biomarkers for several diseases. For testicular germ cell tumors (GCT), circulating microRNAs 371a-3p and 372-3p in serum and plasma have been proposed as biomarkers for diagnostic and disease monitoring purposes. The most widely used method for quantification of specific microRNAs in serum and plasma is reverse transcriptase real-time quantitative PCR (RT-qPCR) by the comparative Ct-method. In this method one or several reference genes or reference microRNAs are needed in order to normalize and calculate the relative microRNA levels across samples. One of the pitfalls in analysis of microRNAs from serum and plasma is the release of microRNAs from blood cells during hemolysis. This is an important issue because varying degrees of hemolysis are not uncommon in routine blood sampling. Thus, hemolysis must be taken into consideration when working with circulating microRNAs from blood. miR-93-5p, miR-30b-5p, and miR-20a-5p have been reported as reference microRNA in analysis of the miR-371a-373 cluster. We here show how these three microRNAs are influenced by hemolysis. We also propose a new reference microRNA, miR-191-5p, which is relatively stable in serum samples with mild hemolysis. In addition, we show how hemolysis can have effect on the reported microRNA levels in patient samples when these reference microRNAs are used in samples with varying levels of hemolysis.https://www.frontiersin.org/article/10.3389/fgene.2019.00463/fullcirculating microRNAserumRT-qPCRtesticular cancerhemolysisquality control |
| spellingShingle | Mette Pernille Myklebust Benedikte Rosenlund Peder Gjengstø Bogdan Stefan Bercea Ása Karlsdottir Marianne Brydøy Olav Dahl Olav Dahl Quantitative PCR Measurement of miR-371a-3p and miR-372-p Is Influenced by Hemolysis Frontiers in Genetics circulating microRNA serum RT-qPCR testicular cancer hemolysis quality control |
| title | Quantitative PCR Measurement of miR-371a-3p and miR-372-p Is Influenced by Hemolysis |
| title_full | Quantitative PCR Measurement of miR-371a-3p and miR-372-p Is Influenced by Hemolysis |
| title_fullStr | Quantitative PCR Measurement of miR-371a-3p and miR-372-p Is Influenced by Hemolysis |
| title_full_unstemmed | Quantitative PCR Measurement of miR-371a-3p and miR-372-p Is Influenced by Hemolysis |
| title_short | Quantitative PCR Measurement of miR-371a-3p and miR-372-p Is Influenced by Hemolysis |
| title_sort | quantitative pcr measurement of mir 371a 3p and mir 372 p is influenced by hemolysis |
| topic | circulating microRNA serum RT-qPCR testicular cancer hemolysis quality control |
| url | https://www.frontiersin.org/article/10.3389/fgene.2019.00463/full |
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