Selection and validation of reference genes for transcript normalization in gene expression studies in Catharanthus roseus

J. Pollier, R. Vanden Bossche, Heiko Rischer, A. Goossens (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

18 Citations (Scopus)

Abstract

Quantitative Real-Time PCR (qPCR), a sensitive and commonly used technique for gene expression analysis, requires stably expressed reference genes for normalization of gene expression. Up to now, only one reference gene for qPCR analysis, corresponding to 40S Ribosomal protein S9 (RPS9), was available for the medicinal plant Catharanthus roseus, the only source of the commercial anticancer drugs vinblastine and vincristine. Here, we screened for additional reference genes for this plant species by mining C.roseus RNA-Seq data for orthologs of 22 genes known to be stably expressed in Arabidopsis thaliana and qualified as superior reference genes for this model plant species. Based on this, eight candidate C.roseus reference genes were identified and, together with RPS9, evaluated by performing qPCR on a series of different C.roseus explants and tissue cultures. NormFinder, geNorm and BestKeeper analyses of the resulting qPCR data revealed that the orthologs of At2g28390 (SAND family protein, SAND), At2g32170 (N2227-like family protein, N2227) and At4g26410 (Expressed protein, EXP) had the highest expression stability across the different C.roseus samples and are superior as reference genes as compared to the traditionally used RPS9. Analysis of publicly available C.roseus RNA-Seq data confirmed the expression stability of SAND and N2227, underscoring their value as reference genes for C.roseus qPCR analysis.
Original languageEnglish
Pages (from-to)20-25
Number of pages6
JournalPlant Physiology and Biochemistry
Volume83
DOIs
Publication statusPublished - 2014
MoE publication typeA1 Journal article-refereed

Fingerprint

Catharanthus
Catharanthus roseus
Gene Expression
gene expression
Genes
Plant Genes
ribosomal proteins
genes
RNA
Proteins
Vinblastine
Vincristine
Medicinal Plants
vinblastine
Arabidopsis
vincristine
proteins
Real-Time Polymerase Chain Reaction
antineoplastic agents
Reference Values

Keywords

  • Catharanthus roseus
  • Madagascar periwinkles
  • reference genes
  • quantative real-time polymerase chain reactions (qPCR)
  • geNorm
  • BestKeeper
  • NormFinder
  • transcript normalisation

Cite this

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title = "Selection and validation of reference genes for transcript normalization in gene expression studies in Catharanthus roseus",
abstract = "Quantitative Real-Time PCR (qPCR), a sensitive and commonly used technique for gene expression analysis, requires stably expressed reference genes for normalization of gene expression. Up to now, only one reference gene for qPCR analysis, corresponding to 40S Ribosomal protein S9 (RPS9), was available for the medicinal plant Catharanthus roseus, the only source of the commercial anticancer drugs vinblastine and vincristine. Here, we screened for additional reference genes for this plant species by mining C.roseus RNA-Seq data for orthologs of 22 genes known to be stably expressed in Arabidopsis thaliana and qualified as superior reference genes for this model plant species. Based on this, eight candidate C.roseus reference genes were identified and, together with RPS9, evaluated by performing qPCR on a series of different C.roseus explants and tissue cultures. NormFinder, geNorm and BestKeeper analyses of the resulting qPCR data revealed that the orthologs of At2g28390 (SAND family protein, SAND), At2g32170 (N2227-like family protein, N2227) and At4g26410 (Expressed protein, EXP) had the highest expression stability across the different C.roseus samples and are superior as reference genes as compared to the traditionally used RPS9. Analysis of publicly available C.roseus RNA-Seq data confirmed the expression stability of SAND and N2227, underscoring their value as reference genes for C.roseus qPCR analysis.",
keywords = "Catharanthus roseus, Madagascar periwinkles, reference genes, quantative real-time polymerase chain reactions (qPCR), geNorm, BestKeeper, NormFinder, transcript normalisation",
author = "J. Pollier and {Vanden Bossche}, R. and Heiko Rischer and A. Goossens",
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Selection and validation of reference genes for transcript normalization in gene expression studies in Catharanthus roseus. / Pollier, J.; Vanden Bossche, R.; Rischer, Heiko; Goossens, A. (Corresponding Author).

In: Plant Physiology and Biochemistry, Vol. 83, 2014, p. 20-25.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Selection and validation of reference genes for transcript normalization in gene expression studies in Catharanthus roseus

AU - Pollier, J.

AU - Vanden Bossche, R.

AU - Rischer, Heiko

AU - Goossens, A.

PY - 2014

Y1 - 2014

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AB - Quantitative Real-Time PCR (qPCR), a sensitive and commonly used technique for gene expression analysis, requires stably expressed reference genes for normalization of gene expression. Up to now, only one reference gene for qPCR analysis, corresponding to 40S Ribosomal protein S9 (RPS9), was available for the medicinal plant Catharanthus roseus, the only source of the commercial anticancer drugs vinblastine and vincristine. Here, we screened for additional reference genes for this plant species by mining C.roseus RNA-Seq data for orthologs of 22 genes known to be stably expressed in Arabidopsis thaliana and qualified as superior reference genes for this model plant species. Based on this, eight candidate C.roseus reference genes were identified and, together with RPS9, evaluated by performing qPCR on a series of different C.roseus explants and tissue cultures. NormFinder, geNorm and BestKeeper analyses of the resulting qPCR data revealed that the orthologs of At2g28390 (SAND family protein, SAND), At2g32170 (N2227-like family protein, N2227) and At4g26410 (Expressed protein, EXP) had the highest expression stability across the different C.roseus samples and are superior as reference genes as compared to the traditionally used RPS9. Analysis of publicly available C.roseus RNA-Seq data confirmed the expression stability of SAND and N2227, underscoring their value as reference genes for C.roseus qPCR analysis.

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