Identification of extremely GC-rich micro RNAs for RT-qPCR data normalization in human plasma
We aimed at extending the repertoire of high-quality miRNA normalizers for reverse transcription-quantitative PCR (RT-qPCR) of human plasma with special emphasis on the extremely guanine-cytosine-rich portion of the miRNome. For high-throughput selection of stable candidates, microarray technology w...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-01-01
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| Series: | Frontiers in Genetics |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fgene.2022.1058668/full |
| _version_ | 1797816698653376512 |
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| author | Volker Baumann Angelos-Theodoros Athanasiou Omid R. Faridani Omid R. Faridani Andreas R. Schwerdtfeger Bernard Wallner Ralf Steinborn Ralf Steinborn |
| author_facet | Volker Baumann Angelos-Theodoros Athanasiou Omid R. Faridani Omid R. Faridani Andreas R. Schwerdtfeger Bernard Wallner Ralf Steinborn Ralf Steinborn |
| author_sort | Volker Baumann |
| collection | DOAJ |
| description | We aimed at extending the repertoire of high-quality miRNA normalizers for reverse transcription-quantitative PCR (RT-qPCR) of human plasma with special emphasis on the extremely guanine-cytosine-rich portion of the miRNome. For high-throughput selection of stable candidates, microarray technology was preferred over small-RNA sequencing (sRNA-seq) since the latter underrepresented miRNAs with a guanine-cytosine (GC) content of at least 75% (p = 0.0002, n = 2). miRNA abundances measured on the microarray were ranked for consistency and uniformity using nine normalization approaches. The eleven most stable sequences included miRNAs of moderate, but also extreme GC content (45%–65%: miR-320d, miR-425-5p, miR-185-5p, miR-486-5p; 80%–95%: miR-1915-3p, miR-3656-5p, miR-3665-5p, miR-3960-5p, miR-4488-5p, miR-4497 and miR-4787-5p). In contrast, the seven extremely GC-rich miRNAs were not found in the two plasma miRNomes screened by sRNA-seq. Stem-loop RT-qPCR was employed for stability verification in 32 plasma samples of healthy male Caucasians (age range: 18–55 years). The lowest inter-individual variance of miRNA abundance was determined for miR-3665 and miR-1915-3p [coefficient of variation (CV) values: 0.08 and 0.50, respectively]. The eight most stable sequences included four extremely GC-rich miRNAs (miR-1915-3p, miR-3665, miR-4787-5p and miR-4497). The best-performing duo normalization factor (NF) for the condition of human plasma, miR-320d and miR-4787-5p, also included a GC-extreme miRNA. In summary, the identification of extremely guanine-cytosine-rich plasma normalizers will help to increase accuracy of PCR-based miRNA quantification, thus raise the potential that miRNAs become markers for psychological stress reactions or early and precise diagnosis of clinical phenotypes. The novel miRNAs might also be useful for orthologous contexts considering their conservation in related animal genomes. |
| first_indexed | 2024-03-13T08:41:34Z |
| format | Article |
| id | doaj.art-e9532f24c23245c48a934ac105a4fe27 |
| institution | Directory Open Access Journal |
| issn | 1664-8021 |
| language | English |
| last_indexed | 2024-03-13T08:41:34Z |
| publishDate | 2023-01-01 |
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| series | Frontiers in Genetics |
| spelling | doaj.art-e9532f24c23245c48a934ac105a4fe272023-05-30T10:44:25ZengFrontiers Media S.A.Frontiers in Genetics1664-80212023-01-011310.3389/fgene.2022.10586681058668Identification of extremely GC-rich micro RNAs for RT-qPCR data normalization in human plasmaVolker Baumann0Angelos-Theodoros Athanasiou1Omid R. Faridani2Omid R. Faridani3Andreas R. Schwerdtfeger4Bernard Wallner5Ralf Steinborn6Ralf Steinborn7Genomics Core Facility, VetCore, University of Veterinary Medicine, Vienna, AustriaGenomics Core Facility, VetCore, University of Veterinary Medicine, Vienna, AustriaGarvan Institute of Medical Research, Sydney, NSW, AustraliaLowy Cancer Research Centre, School of Biomedical Sciences, University of New South Wales, Sydney, NSW, AustraliaInstitute of Psychology, Karl-Franzens-University Graz, Graz, AustriaDepartment of Behavioral and Cognitive Biology, University of Vienna, Vienna, AustriaGenomics Core Facility, VetCore, University of Veterinary Medicine, Vienna, AustriaDepartment of Microbiology, Immunobiology and Genetics, University of Vienna, Vienna, AustriaWe aimed at extending the repertoire of high-quality miRNA normalizers for reverse transcription-quantitative PCR (RT-qPCR) of human plasma with special emphasis on the extremely guanine-cytosine-rich portion of the miRNome. For high-throughput selection of stable candidates, microarray technology was preferred over small-RNA sequencing (sRNA-seq) since the latter underrepresented miRNAs with a guanine-cytosine (GC) content of at least 75% (p = 0.0002, n = 2). miRNA abundances measured on the microarray were ranked for consistency and uniformity using nine normalization approaches. The eleven most stable sequences included miRNAs of moderate, but also extreme GC content (45%–65%: miR-320d, miR-425-5p, miR-185-5p, miR-486-5p; 80%–95%: miR-1915-3p, miR-3656-5p, miR-3665-5p, miR-3960-5p, miR-4488-5p, miR-4497 and miR-4787-5p). In contrast, the seven extremely GC-rich miRNAs were not found in the two plasma miRNomes screened by sRNA-seq. Stem-loop RT-qPCR was employed for stability verification in 32 plasma samples of healthy male Caucasians (age range: 18–55 years). The lowest inter-individual variance of miRNA abundance was determined for miR-3665 and miR-1915-3p [coefficient of variation (CV) values: 0.08 and 0.50, respectively]. The eight most stable sequences included four extremely GC-rich miRNAs (miR-1915-3p, miR-3665, miR-4787-5p and miR-4497). The best-performing duo normalization factor (NF) for the condition of human plasma, miR-320d and miR-4787-5p, also included a GC-extreme miRNA. In summary, the identification of extremely guanine-cytosine-rich plasma normalizers will help to increase accuracy of PCR-based miRNA quantification, thus raise the potential that miRNAs become markers for psychological stress reactions or early and precise diagnosis of clinical phenotypes. The novel miRNAs might also be useful for orthologous contexts considering their conservation in related animal genomes.https://www.frontiersin.org/articles/10.3389/fgene.2022.1058668/fullmiRNA expression microarraysmall-RNA sequencingstem-loop reverse-transcription quantitative PCRhuman plasma miRNAsmiRNA reference genescognitive stress-coping |
| spellingShingle | Volker Baumann Angelos-Theodoros Athanasiou Omid R. Faridani Omid R. Faridani Andreas R. Schwerdtfeger Bernard Wallner Ralf Steinborn Ralf Steinborn Identification of extremely GC-rich micro RNAs for RT-qPCR data normalization in human plasma Frontiers in Genetics miRNA expression microarray small-RNA sequencing stem-loop reverse-transcription quantitative PCR human plasma miRNAs miRNA reference genes cognitive stress-coping |
| title | Identification of extremely GC-rich micro RNAs for RT-qPCR data normalization in human plasma |
| title_full | Identification of extremely GC-rich micro RNAs for RT-qPCR data normalization in human plasma |
| title_fullStr | Identification of extremely GC-rich micro RNAs for RT-qPCR data normalization in human plasma |
| title_full_unstemmed | Identification of extremely GC-rich micro RNAs for RT-qPCR data normalization in human plasma |
| title_short | Identification of extremely GC-rich micro RNAs for RT-qPCR data normalization in human plasma |
| title_sort | identification of extremely gc rich micro rnas for rt qpcr data normalization in human plasma |
| topic | miRNA expression microarray small-RNA sequencing stem-loop reverse-transcription quantitative PCR human plasma miRNAs miRNA reference genes cognitive stress-coping |
| url | https://www.frontiersin.org/articles/10.3389/fgene.2022.1058668/full |
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