title: Identification and evaluation of reference genes for qRT-PCR normalization in Ganoderma lucidum.
abstract: Quantitative real-time reverse transcription PCR (qRT-PCR) is a rapid, sensitive, and reliable technique for gene expression studies. The accuracy and reliability of qRT-PCR results depend on the stability of the reference genes used for gene normalization. Therefore, a systematic process of reference gene evaluation is needed. Ganoderma lucidum is a famous medicinal mushroom in East Asia. In the current study, 10 potential reference genes were selected from the G. lucidum genomic data. The sequences of these genes were manually curated, and primers were designed following strict criteria. The experiment was conducted using qRT-PCR, and the stability of each candidate gene was assessed using four commonly used statistical programs-geNorm, NormFinder, BestKeeper, and RefFinder. According to our results, PP2A was expressed at the most stable levels under different fermentation conditions, and RPL4 was the most stably expressed gene in different tissues. RPL4, PP2A, and β-tubulin are the most commonly recommended reference genes for normalizing gene expression in the entire sample set. The current study provides a foundation for the further use of qRT-PCR in G. lucidum gene analysis.
authors.affiliation: The National Engineering Laboratory for Breeding of Endangered Medicinal Materials, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 151, Malianwa North Road, HaiDian District, Beijing, 100193, China, xj8442cn@gmail.com.
authors.forename: Jiang
authors.initials: J
authors.lastname: Xu
authors.forename: ZhiChao
authors.initials: Z
authors.lastname: Xu
authors.forename: YingJie
authors.initials: Y
authors.lastname: Zhu
authors.forename: HongMei
authors.initials: H
authors.lastname: Luo
authors.forename: Jun
authors.initials: J
authors.lastname: Qian
authors.forename: AiJia
authors.initials: A
authors.lastname: Ji
authors.forename: YuanLei
authors.initials: Y
authors.lastname: Hu
authors.forename: Wei
authors.initials: W
authors.lastname: Sun
authors.forename: Bo
authors.initials: B
authors.lastname: Wang
authors.forename: JingYuan
authors.initials: J
authors.lastname: Song
authors.forename: Chao
authors.initials: C
authors.lastname: Sun
authors.forename: ShiLin
authors.initials: S
authors.lastname: Chen
mesh_terms: D015870:Gene Expression; D005800:Genes, Fungal / Q000235:genetics*; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012015:Reference Standards; D020940:Reishi / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000592:standards*
chemical_list: D012331:RNA, Fungal