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Article ; Online: Artifacts and biases of the reverse transcription reaction in RNA sequencing.

Verwilt, Jasper / Mestdagh, Pieter / Vandesompele, Jo

2023 Volume 29, Issue 7, Page(s) 889–897

Abstract: RNA sequencing has spurred a significant number of research areas in recent years. Most protocols rely on synthesizing a more stable complementary DNA (cDNA) copy of the RNA molecule during the reverse transcription reaction. The resulting cDNA pool is ... ...

Abstract	RNA sequencing has spurred a significant number of research areas in recent years. Most protocols rely on synthesizing a more stable complementary DNA (cDNA) copy of the RNA molecule during the reverse transcription reaction. The resulting cDNA pool is often wrongfully assumed to be quantitatively and molecularly similar to the original RNA input. Sadly, biases and artifacts confound the resulting cDNA mixture. These issues are often overlooked or ignored in the literature by those that rely on the reverse transcription process. In this review, we confront the reader with intra- and intersample biases and artifacts caused by the reverse transcription reaction during RNA sequencing experiments. To fight the reader's despair, we also provide solutions to most issues and inform on good RNA sequencing practices. We hope the reader can use this review to their advantage, thereby contributing to scientifically sound RNA studies.
MeSH term(s)	Reverse Transcription ; DNA, Complementary/genetics ; Artifacts ; RNA/genetics ; Sequence Analysis, RNA/methods ; Bias
Chemical Substances	DNA, Complementary ; RNA (63231-63-0)
Language	English
Publishing date	2023-03-29
Publishing country	United States
Document type	Review ; Journal Article
ZDB-ID	1241540-6
ISSN	1469-9001 ; 1355-8382
ISSN (online)	1469-9001
ISSN	1355-8382
DOI	10.1261/rna.079623.123
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Article ; Online: Evaluation of efficiency and sensitivity of 1D and 2D sample pooling strategies for SARS-CoV-2 RT-qPCR screening purposes.

Verwilt, Jasper / Hellemans, Jan / Sante, Tom / Mestdagh, Pieter / Vandesompele, Jo

Scientific reports

2022 Volume 12, Issue 1, Page(s) 6603

Abstract: To increase the throughput, lower the cost, and save scarce test reagents, laboratories can pool patient samples before SARS-CoV-2 RT-qPCR testing. While different sample pooling methods have been proposed and effectively implemented in some laboratories, ...

Abstract	To increase the throughput, lower the cost, and save scarce test reagents, laboratories can pool patient samples before SARS-CoV-2 RT-qPCR testing. While different sample pooling methods have been proposed and effectively implemented in some laboratories, no systematic and large-scale evaluations exist using real-life quantitative data gathered throughout the different epidemiological stages. Here, we use anonymous data from 9673 positive cases to model, simulate and compare 1D and 2D pooling strategies. We show that the optimal choice of pooling method and pool size is an intricate decision with a testing population-dependent efficiency-sensitivity trade-off and present an online tool to provide the reader with custom real-time 1D pooling strategy recommendations.
MeSH term(s)	COVID-19/diagnosis ; COVID-19 Nucleic Acid Testing ; COVID-19 Testing ; Humans ; Real-Time Polymerase Chain Reaction/methods ; SARS-CoV-2/genetics ; Sensitivity and Specificity ; Specimen Handling/methods
Language	English
Publishing date	2022-04-22
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-022-10581-6
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Article: Circulating microRNAs as Non-Invasive Biomarkers in Endometriosis Diagnosis-A Systematic Review.

Vanhie, Arne / Caron, Ellen / Vermeersch, Eveline / O, Dorien / Tomassetti, Carla / Meuleman, Christel / Mestdagh, Pieter / D'Hooghe, Thomas M

Biomedicines

2024 Volume 12, Issue 4

Abstract: The aim of this systematic review is to assess the power of circulating miRNAs as biomarkers as a diagnostic tool in endometriosis. In endometriosis-suspected women with uncertain imaging, the only way to confirm or exclude endometriosis with certainty ... ...

Abstract	The aim of this systematic review is to assess the power of circulating miRNAs as biomarkers as a diagnostic tool in endometriosis. In endometriosis-suspected women with uncertain imaging, the only way to confirm or exclude endometriosis with certainty is currently laparoscopy. This creates a need for non-invasive diagnostics. We searched the literature through the PubMed database using the Mesh terms 'endometriosis' and 'miRNAs'. Some, but limited, overlap was found between the 32 articles included, with a total of 20 miRNAs reported as dysregulated in endometriosis in two or more studies. MiR-17-5p was reported as dysregulated in six studies, followed by miR-451a and let-7b-5p in four studies and miR-20a-5p, miR-143-3p, miR-199a-5p and miR-3613-5p in three studies. Furthermore, a possible impact of the menstrual phase on miRNA expression was noted in five studies, while no influence of hormonal intake was observed in any included study. The modest reproducibility between studies may be attributable to biological variability as well as to the lack of universal protocols, resulting in pre- and analytical variability. Despite the identification of several suitable candidate biomarkers among the miRNAs, the need for high-quality studies with larger and well-defined population cohorts and the use of standardized protocols lingers.
Language	English
Publishing date	2024-04-17
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2720867-9
ISSN	2227-9059
ISSN	2227-9059
DOI	10.3390/biomedicines12040888
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Article ; Online: CiLiQuant: Quantification of RNA Junction Reads Based on Their Circular or Linear Transcript Origin.

Morlion, Annelien / Hulstaert, Eva / Vromman, Marieke / Anckaert, Jasper / Everaert, Celine / Vandesompele, Jo / Mestdagh, Pieter

Frontiers in bioinformatics

2022 Volume 2, Page(s) 834034

Abstract: Distinguishing circular RNA reads from reads derived from the linear host transcript is a challenging task because of sequence overlap. We developed a computational approach, CiLiQuant, that determines the relative circular and linear abundance of ... ...

Abstract	Distinguishing circular RNA reads from reads derived from the linear host transcript is a challenging task because of sequence overlap. We developed a computational approach, CiLiQuant, that determines the relative circular and linear abundance of transcripts and gene loci using back-splice and unambiguous forward-splice junction reads generated by existing mapping and circular RNA discovery tools.
Language	English
Publishing date	2022-02-22
Publishing country	Switzerland
Document type	Journal Article
ISSN	2673-7647
ISSN (online)	2673-7647
DOI	10.3389/fbinf.2022.834034
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Article ; Online: Unlocking the secrets of long non-coding RNAs in asthma.

Gysens, Fien / Mestdagh, Pieter / de Bony de Lavergne, Eric / Maes, Tania

Thorax

2022 Volume 77, Issue 5, Page(s) 514–522

Abstract: Asthma is a very heterozygous disease, divided in subtypes, such as eosinophilic and neutrophilic asthma. Phenotyping and endotyping of patients, especially patients with severe asthma who are refractory to standard treatment, are crucial in asthma ... ...

Abstract	Asthma is a very heterozygous disease, divided in subtypes, such as eosinophilic and neutrophilic asthma. Phenotyping and endotyping of patients, especially patients with severe asthma who are refractory to standard treatment, are crucial in asthma management and are based on a combination of clinical and biological features. Nevertheless, the quest remains to find better biomarkers that distinguish asthma subtypes in a more clear and objective manner and to find new therapeutic targets to treat people with therapy-resistant asthma. In the past, research to identify asthma subtypes mainly focused on expression profiles of protein-coding genes. However, advances in RNA-sequencing technologies and the discovery of non-coding RNAs as important post-transcriptional regulators have provided an entire new field of research opportunities in asthma. This review focusses on long non-coding RNAs (lncRNAs) in asthma; these are non-coding RNAs with a length of more than 200 nucleotides. Many lncRNAs are differentially expressed in asthma, and several have been associated with asthma severity or inflammatory phenotype. Moreover,
MeSH term(s)	Asthma/genetics ; Asthma/metabolism ; Biomarkers ; Humans ; RNA, Long Noncoding/genetics ; RNA, Long Noncoding/metabolism ; Sequence Analysis, RNA
Chemical Substances	Biomarkers ; RNA, Long Noncoding
Language	English
Publishing date	2022-03-04
Publishing country	England
Document type	Journal Article ; Review ; Research Support, Non-U.S. Gov't
ZDB-ID	204353-1
ISSN	1468-3296 ; 0040-6376
ISSN (online)	1468-3296
ISSN	0040-6376
DOI	10.1136/thoraxjnl-2021-218359
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Article ; Online: Direct lysis of 3D cell cultures for RT-qPCR gene expression quantification.

Gysens, Fien / Ostyn, Lisa / Goeteyn, Ellen / Blondeel, Eva / Nuyttens, Justine / De Wever, Olivier / de Bony, Eric / Crabbé, Aurélie / Mestdagh, Pieter

Scientific reports

2023 Volume 13, Issue 1, Page(s) 1520

Abstract: In vitro cell culture experiments are widely used to study cellular behavior in most biological research fields. Except for suspension cells, most human cell types are cultured as adherent monolayers on a plastic surface. While technically convenient, ... ...

Abstract	In vitro cell culture experiments are widely used to study cellular behavior in most biological research fields. Except for suspension cells, most human cell types are cultured as adherent monolayers on a plastic surface. While technically convenient, monolayer cultures can suffer from limitations in terms of physiological relevance, as their resemblance to complex in vivo tissue structures is limited. To address these limitations, three-dimensional (3D) cell culture systems have gained increased interest as they mimic key structural and functional properties of their in vivo tissue counterparts. Nevertheless, protocols established on monolayer cell cultures may require adjustments if they are to be applied to 3D cell cultures. As gene expression quantification is an essential part of many in vitro experiments, we evaluated and optimized a direct cell lysis, reverse transcription and qPCR protocol applicable for 3D cell cultures. The newly developed protocol wherein gene expression is determined directly from crude cell lysates showed improved cell lysis compared to the standard protocol, accurate gene expression quantification, hereby avoiding time-consuming cell harvesting and RNA extraction.
MeSH term(s)	Humans ; Cell Culture Techniques/methods ; Cell Culture Techniques, Three Dimensional ; Gene Expression
Language	English
Publishing date	2023-01-27
Publishing country	England
Document type	Journal Article
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-023-28844-1
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Article: Blocking Abundant RNA Transcripts by High-Affinity Oligonucleotides during Transcriptome Library Preparation.

Everaert, Celine / Verwilt, Jasper / Verniers, Kimberly / Vandamme, Niels / Marcos Rubio, Alvaro / Vandesompele, Jo / Mestdagh, Pieter

Biological procedures online

2023 Volume 25, Issue 1, Page(s) 7

Abstract: Background: RNA sequencing has become the gold standard for transcriptome analysis but has an inherent limitation of challenging quantification of low-abundant transcripts. In contrast to microarray technology, RNA sequencing reads are proportionally ... ...

Abstract	Background: RNA sequencing has become the gold standard for transcriptome analysis but has an inherent limitation of challenging quantification of low-abundant transcripts. In contrast to microarray technology, RNA sequencing reads are proportionally divided in function of transcript abundance. Therefore, low-abundant RNAs compete against highly abundant - and sometimes non-informative - RNA species. Results: We developed an easy-to-use strategy based on high-affinity RNA-binding oligonucleotides to block reverse transcription and PCR amplification of specific RNA transcripts, thereby substantially reducing their abundance in the final sequencing library. To demonstrate the broad application potential of our method, we applied it to different transcripts and library preparation strategies, including YRNAs in small RNA sequencing of human blood plasma, mitochondrial rRNAs in both 3' end sequencing and long-read sequencing, and MALAT1 in single-cell 3' end sequencing. We demonstrate that the blocking strategy is highly efficient, reproducible, specific, and generally results in better transcriptome coverage and complexity. Conclusion: Our method does not require modifications of the library preparation procedure apart from simply adding blocking oligonucleotides to the RT reaction and can thus be easily integrated into virtually any RNA sequencing library preparation protocol.
Language	English
Publishing date	2023-03-08
Publishing country	England
Document type	Journal Article
ZDB-ID	2027823-8
ISSN	1480-9222
ISSN	1480-9222
DOI	10.1186/s12575-023-00193-3
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Article ; Online: MicroRNAs as future therapeutic targets in COPD?

Bracke, Ken R / Mestdagh, Pieter

The European respiratory journal

2017 Volume 49, Issue 5

MeSH term(s)	Humans ; MicroRNAs ; Pulmonary Disease, Chronic Obstructive
Chemical Substances	MicroRNAs
Language	English
Publishing date	2017
Publishing country	England
Document type	Editorial ; Comment
ZDB-ID	639359-7
ISSN	1399-3003 ; 0903-1936
ISSN (online)	1399-3003
ISSN	0903-1936
DOI	10.1183/13993003.00431-2017
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Zs.A 2322: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (2.OG) ab Jg. 2022: Lesesaal (EG)
Zs.MO 292: Show issues

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Article ; Online: Custom long non-coding RNA capture enhances detection sensitivity in different human sample types

Morlion, Annelien / Everaert, Celine / Nuytens, Justine / Hulstaert, Eva / Vandesompele, Jo / Mestdagh, Pieter

RNA Biology. 2021 Oct. 15, v. 18, no. S1 p.215-222

2021

Abstract: Long non-coding RNAs (lncRNAs) are a heterogeneous group of transcripts that lack protein coding potential and display regulatory functions in various cellular processes. As a result of their cell- and cancer-specific expression patterns, lncRNAs have ... ...

Abstract	Long non-coding RNAs (lncRNAs) are a heterogeneous group of transcripts that lack protein coding potential and display regulatory functions in various cellular processes. As a result of their cell- and cancer-specific expression patterns, lncRNAs have emerged as potential diagnostic and therapeutic targets. The accurate characterization of lncRNAs in bulk transcriptome data remains challenging due to their low abundance compared to protein coding genes. To tackle this issue, we describe a unique short-read custom lncRNA capture sequencing approach that relies on a comprehensive set of 565,878 capture probes for 49,372 human lncRNA genes. This custom lncRNA capture approach was evaluated on various sample types ranging from artificial high-quality RNA mixtures to more challenging formalin-fixed paraffin-embedded tissue and biofluid material. The custom enrichment approach allows the detection of a more diverse repertoire of lncRNAs, with better reproducibility and higher coverage compared to classic total RNA-sequencing.
Keywords	detection limit ; humans ; non-coding RNA ; sequence analysis ; therapeutics ; transcriptome ; lncRNA ; RNA sequencing ; probes ; lncRNome ; RNA abundance ; RNA expression ; FFPE ; biofluid
Language	English
Dates of publication	2021-1015
Size	p. 215-222.
Publishing place	Taylor & Francis
Document type	Article ; Online
ZDB-ID	2159587-2
ISSN	1555-8584
ISSN	1555-8584
DOI	10.1080/15476286.2021.1971438
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Candidate RNA biomarkers in biofluids for early diagnosis of ovarian cancer: A systematic review.

Hulstaert, Eva / Morlion, Annelien / Levanon, Keren / Vandesompele, Jo / Mestdagh, Pieter

Gynecologic oncology

2020 Volume 160, Issue 2, Page(s) 633–642

Abstract: Ovarian cancer is often diagnosed in an advanced stage and is associated with a high mortality rate. It is assumed that early detection of ovarian cancer could improve patient outcomes. Unfortunately, effective screening methods for early diagnosis of ... ...

Abstract	Ovarian cancer is often diagnosed in an advanced stage and is associated with a high mortality rate. It is assumed that early detection of ovarian cancer could improve patient outcomes. Unfortunately, effective screening methods for early diagnosis of ovarian cancer are still lacking. Extracellular RNAs circulating in human biofluids can reliably be measured and are emerging as potential biomarkers in cancer. In this systematic review, we present 75 RNA biomarkers detectable in human biofluids that have been studied for early diagnosis of ovarian cancer. The majority of these markers are microRNAs identified using RT-qPCR or microarrays in blood-based fluids. A handful of studies used RNA-sequencing and explored alternative fluids, such as urine and ascites. Candidate RNA biomarkers that were more abundant in biofluids of ovarian cancer patients compared to controls in at least two independent studies include miR-21, the miR-200 family, miR-205, miR-10a and miR-346. Amongst the markers confirmed to be lower in at least two studies are miR-122, miR-193a, miR-223, miR-126 and miR-106b. While these biomarkers show promising diagnostic potential, further validation is required before implementation in routine clinical care. Challenges related to biomarker validation and reflections on future perspectives to accelerate progress in this field are discussed.
MeSH term(s)	Ascitic Fluid/chemistry ; Ascitic Fluid/pathology ; Biomarkers, Tumor/analysis ; Biomarkers, Tumor/genetics ; Carcinoma, Ovarian Epithelial/blood ; Carcinoma, Ovarian Epithelial/diagnosis ; Carcinoma, Ovarian Epithelial/genetics ; Carcinoma, Ovarian Epithelial/urine ; Early Detection of Cancer/methods ; Female ; Humans ; Liquid Biopsy/methods ; MicroRNAs/analysis ; MicroRNAs/genetics ; Ovarian Neoplasms/blood ; Ovarian Neoplasms/diagnosis ; Ovarian Neoplasms/genetics ; Ovarian Neoplasms/urine ; RNA-Seq
Chemical Substances	Biomarkers, Tumor ; MicroRNAs
Language	English
Publishing date	2020-11-27
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Systematic Review
ZDB-ID	801461-9
ISSN	1095-6859 ; 0090-8258
ISSN (online)	1095-6859
ISSN	0090-8258
DOI	10.1016/j.ygyno.2020.11.018
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