mRNA extraction of Xanthomonas fragariae in strawberry and validation of reference genes for the RT-qPCR for study of bacterial gene expression.
Gene expression
RNA isolation
RT-qPCR
Reference genes
Xanthomonas fragariae
Journal
Molecular biology reports
ISSN: 1573-4978
Titre abrégé: Mol Biol Rep
Pays: Netherlands
ID NLM: 0403234
Informations de publication
Date de publication:
Dec 2019
Dec 2019
Historique:
received:
29
03
2019
accepted:
26
07
2019
pubmed:
2
8
2019
medline:
2
5
2020
entrez:
2
8
2019
Statut:
ppublish
Résumé
This is the first study which describes a unique procedure of isolating of high-quality, intact RNA from strawberry leaves of Xanthomonas fragariae, three most suitable reference genes, crucial for the normalization of RT-qPCR data for this pathogen and accurate expression analysis of target genes. In our study, various mathematic algorithms: NormFinder geNorm, BestKeeper, the delta CT method, RefFinder were adopted for validation of most stable reference genes from nine candidate genes (ffh, glyA, gyrA, gyrB, proC, pykA, recA, rpoB, rpoD). The analyses allowing to select three most suitable pioneer reference genes, gyrB, ffh, and pykA, that we recommend for the normalization of RT-qPCR data and for the study of the expression of target genes in Xf. Moreover, their combination as references allowed for an accurate expression analysis and computation of the fold change of the flhF and iroN2 genes in Xf. These two genes are important for the success of the colonization of plant tissue and pathogenicity and sequences of primers designed to study these genes, are presented.
Identifiants
pubmed: 31368022
doi: 10.1007/s11033-019-05006-z
pii: 10.1007/s11033-019-05006-z
doi:
Substances chimiques
RNA, Bacterial
0
RNA, Messenger
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
5723-5733Subventions
Organisme : Ministerstwo Nauki i Szkolnictwa Wyższego
ID : project 3195/7.PR/2014/2
Organisme : European Union Seventh Programme
ID : FP7/2007-2013
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