Identification of valid reference genes for microRNA expression studies in a hepatitis B virus replicating liver cell line

Kari Stougaard Jacobsen; Kirstine Overgaard Nielsen; Thilde Nordmann Winther; Dieter Glebe; Flemming Pociot; Birthe Hogh

doi:10.1186/s13104-016-1848-2

Identification of valid reference genes for microRNA expression studies in a hepatitis B virus replicating liver cell line

Kari Stougaard Jacobsen, Kirstine Overgaard Nielsen, Thilde Nordmann Winther, Dieter Glebe, Flemming Pociot, Birthe Hogh

Department of Clinical Medicine

7 Citations (Scopus)

Abstract

BACKGROUND: MicroRNAs are regulatory molecules and suggested as non-invasive biomarkers for molecular diagnostics and prognostics. Altered expression levels of specific microRNAs are associated with hepatitis B virus infection and hepatocellular carcinoma. We previously identified differentially expressed microRNAs with liver-specific target genes in plasma from children with chronic hepatitis B. To further understand the biological role of these microRNAs in the pathogenesis of chronic hepatitis B, we have used the human liver cell line HepG2, with and without HBV replication, after transfection of hepatitis B virus expression vectors. RT-qPCR is the preferred method for microRNA studies, and a careful normalisation strategy, verifying the optimal set of reference genes, is decisive for correctly evaluating microRNA expression levels. The aim of this study was to provide valid reference genes for the human HCC-derived cell line HepG2.

RESULTS: A panel of 739 microRNAs was screened to identify the most stably expressed microRNAs, followed by a PubMed search identifying microRNAs previously used as reference genes. Sixteen candidate reference genes were validated by RT-qPCR. Reference gene stabilities were calculated first by standard deviations of ΔCt values and then by geNorm and NormFinder analyses, taking into account the amplification efficiency of each microRNA primer set. The optimal set of reference genes was verified by a target analysis using RT-qPCR on miR-215-5p.

CONCLUSION: We identified miR-24-3p, miR-151a-5p, and miR-425-5p as the most valid combination of reference genes for microRNA RT-qPCR studies in our hepatitis B virus replicating HepG2 cell model.

Original language	English
Article number	38
Journal	BMC Research Notes
Volume	9
Pages (from-to)	1-7
Number of pages	7
ISSN	1756-0500
DOIs	https://doi.org/10.1186/s13104-016-1848-2
Publication status	Published - 22 Jan 2016

Keywords

Gene Expression Profiling
Gene Expression Regulation, Neoplastic
Genes, Essential
Hep G2 Cells
Hepatitis B virus
Humans
MicroRNAs
Real-Time Polymerase Chain Reaction
Reference Standards
Software
Virus Replication
Journal Article

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1186/s13104-016-1848-2

Cite this

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title = "Identification of valid reference genes for microRNA expression studies in a hepatitis B virus replicating liver cell line",

abstract = "BACKGROUND: MicroRNAs are regulatory molecules and suggested as non-invasive biomarkers for molecular diagnostics and prognostics. Altered expression levels of specific microRNAs are associated with hepatitis B virus infection and hepatocellular carcinoma. We previously identified differentially expressed microRNAs with liver-specific target genes in plasma from children with chronic hepatitis B. To further understand the biological role of these microRNAs in the pathogenesis of chronic hepatitis B, we have used the human liver cell line HepG2, with and without HBV replication, after transfection of hepatitis B virus expression vectors. RT-qPCR is the preferred method for microRNA studies, and a careful normalisation strategy, verifying the optimal set of reference genes, is decisive for correctly evaluating microRNA expression levels. The aim of this study was to provide valid reference genes for the human HCC-derived cell line HepG2.RESULTS: A panel of 739 microRNAs was screened to identify the most stably expressed microRNAs, followed by a PubMed search identifying microRNAs previously used as reference genes. Sixteen candidate reference genes were validated by RT-qPCR. Reference gene stabilities were calculated first by standard deviations of ΔCt values and then by geNorm and NormFinder analyses, taking into account the amplification efficiency of each microRNA primer set. The optimal set of reference genes was verified by a target analysis using RT-qPCR on miR-215-5p.CONCLUSION: We identified miR-24-3p, miR-151a-5p, and miR-425-5p as the most valid combination of reference genes for microRNA RT-qPCR studies in our hepatitis B virus replicating HepG2 cell model.",

keywords = "Gene Expression Profiling, Gene Expression Regulation, Neoplastic, Genes, Essential, Hep G2 Cells, Hepatitis B virus, Humans, MicroRNAs, Real-Time Polymerase Chain Reaction, Reference Standards, Software, Virus Replication, Journal Article",

author = "Jacobsen, {Kari Stougaard} and Nielsen, {Kirstine Overgaard} and {Nordmann Winther}, Thilde and Dieter Glebe and Flemming Pociot and Birthe Hogh",

year = "2016",

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day = "22",

doi = "10.1186/s13104-016-1848-2",

language = "English",

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journal = "BMC Research Notes",

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TY - JOUR

T1 - Identification of valid reference genes for microRNA expression studies in a hepatitis B virus replicating liver cell line

AU - Jacobsen, Kari Stougaard

AU - Nielsen, Kirstine Overgaard

AU - Nordmann Winther, Thilde

AU - Glebe, Dieter

AU - Pociot, Flemming

AU - Hogh, Birthe

PY - 2016/1/22

Y1 - 2016/1/22

N2 - BACKGROUND: MicroRNAs are regulatory molecules and suggested as non-invasive biomarkers for molecular diagnostics and prognostics. Altered expression levels of specific microRNAs are associated with hepatitis B virus infection and hepatocellular carcinoma. We previously identified differentially expressed microRNAs with liver-specific target genes in plasma from children with chronic hepatitis B. To further understand the biological role of these microRNAs in the pathogenesis of chronic hepatitis B, we have used the human liver cell line HepG2, with and without HBV replication, after transfection of hepatitis B virus expression vectors. RT-qPCR is the preferred method for microRNA studies, and a careful normalisation strategy, verifying the optimal set of reference genes, is decisive for correctly evaluating microRNA expression levels. The aim of this study was to provide valid reference genes for the human HCC-derived cell line HepG2.RESULTS: A panel of 739 microRNAs was screened to identify the most stably expressed microRNAs, followed by a PubMed search identifying microRNAs previously used as reference genes. Sixteen candidate reference genes were validated by RT-qPCR. Reference gene stabilities were calculated first by standard deviations of ΔCt values and then by geNorm and NormFinder analyses, taking into account the amplification efficiency of each microRNA primer set. The optimal set of reference genes was verified by a target analysis using RT-qPCR on miR-215-5p.CONCLUSION: We identified miR-24-3p, miR-151a-5p, and miR-425-5p as the most valid combination of reference genes for microRNA RT-qPCR studies in our hepatitis B virus replicating HepG2 cell model.

AB - BACKGROUND: MicroRNAs are regulatory molecules and suggested as non-invasive biomarkers for molecular diagnostics and prognostics. Altered expression levels of specific microRNAs are associated with hepatitis B virus infection and hepatocellular carcinoma. We previously identified differentially expressed microRNAs with liver-specific target genes in plasma from children with chronic hepatitis B. To further understand the biological role of these microRNAs in the pathogenesis of chronic hepatitis B, we have used the human liver cell line HepG2, with and without HBV replication, after transfection of hepatitis B virus expression vectors. RT-qPCR is the preferred method for microRNA studies, and a careful normalisation strategy, verifying the optimal set of reference genes, is decisive for correctly evaluating microRNA expression levels. The aim of this study was to provide valid reference genes for the human HCC-derived cell line HepG2.RESULTS: A panel of 739 microRNAs was screened to identify the most stably expressed microRNAs, followed by a PubMed search identifying microRNAs previously used as reference genes. Sixteen candidate reference genes were validated by RT-qPCR. Reference gene stabilities were calculated first by standard deviations of ΔCt values and then by geNorm and NormFinder analyses, taking into account the amplification efficiency of each microRNA primer set. The optimal set of reference genes was verified by a target analysis using RT-qPCR on miR-215-5p.CONCLUSION: We identified miR-24-3p, miR-151a-5p, and miR-425-5p as the most valid combination of reference genes for microRNA RT-qPCR studies in our hepatitis B virus replicating HepG2 cell model.

KW - Gene Expression Profiling

KW - Gene Expression Regulation, Neoplastic

KW - Genes, Essential

KW - Hep G2 Cells

KW - Hepatitis B virus

KW - Humans

KW - MicroRNAs

KW - Real-Time Polymerase Chain Reaction

KW - Reference Standards

KW - Software

KW - Virus Replication

KW - Journal Article

U2 - 10.1186/s13104-016-1848-2

DO - 10.1186/s13104-016-1848-2

M3 - Journal article

C2 - 26801621

SN - 1756-0500

VL - 9

SP - 1

EP - 7

JO - BMC Research Notes

JF - BMC Research Notes

M1 - 38

ER -

Identification of valid reference genes for microRNA expression studies in a hepatitis B virus replicating liver cell line

Abstract

Keywords

UN SDGs

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