title: Selection and validation of reference genes for normalization of quantitative real-time reverse transcription PCR analysis in Poria cocos (Schw.) Wolf (Fuling).
abstract: BACKGROUND
  Quantitative real-time reverse transcription PCR (qRT-PCR) requires a stable internal control to avoid misinterpretation of data or errors for gene expression normalization. However, there are still no validated reference genes for stable internal control in Poria cocos (Schw.) Wolf (Fuling). This study aims to validate the reference genes of P. cocos.
  
  
  METHODS
  This study firstly collected the 14 candidate reference genes by BLASTP from the genome of P. cocos for qRT-PCR analysis to determine the expression levels of 14 housekeeping genes (GAPDH, MAPK, β-Act, RPB2, RPB1-1, RPB1-2, his3-1, his3-2, APT, SAMDC, RP, β-Tub, EIF, and CYP) under different temperatures and in response to different plant hormones (indole-3-acetic acid, abscisic acid, 6-benzylaminopurine, methyl jasmonate, and gibberellic acid), and the threshold cycle (Ct) values. The results were analyzed by four programs (i.e., geNorm, NormFinder, BestKeeper, and RefFinder) for evaluating the candidate reference genes.
  
  
  RESULTS
  SAMDC, his3-2, RP, RPB2, and his3-1 were recommended as reference genes for treating P. cocos with indole-3-acetic acid, abscisic acid, 6-benzylaminopurine, methyl jasmonate, and gibberellic acid, respectively. Under different temperatures RPB2 was the most stable reference gene. CYP was the most stable gene for all 90 samples by RefFinder.
  
  
  CONCLUSIONS
  SAMDC, his3-2, RP, RPB2, and his3-1 were evaluated to be suitable reference genes for P. cocos following different treatments. RPB2 was the most stable reference gene under different temperatures and CYP was the most stable gene in the mycelia under all six evaluated conditions.
authors.affiliation: Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100193 China.
authors.forename: Xin
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100193 China.
authors.forename: Zhi-Chao
authors.initials: ZC
authors.lastname: Xu
authors.affiliation: Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700 China.
authors.forename: Jiang
authors.initials: J
authors.lastname: Xu
authors.affiliation: Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100193 China.
authors.forename: Ai-Jia
authors.initials: AJ
authors.lastname: Ji
authors.affiliation: Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100193 China.
authors.forename: Hong-Mei
authors.initials: HM
authors.lastname: Luo
authors.affiliation: Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100193 China ; Chongqing Institute of Medicinal Plant Cultivation, Chongqing, 408435 China.
authors.forename: Jing-Yuan
authors.initials: JY
authors.lastname: Song
authors.affiliation: Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100193 China.
authors.forename: Chao
authors.initials: C
authors.lastname: Sun
authors.affiliation: State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, 100871 China.
authors.forename: Yuan-Lei
authors.initials: YL
authors.lastname: Hu
authors.affiliation: Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100193 China ; Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, 100700 China.
authors.forename: Shi-Lin
authors.initials: SL
authors.lastname: Chen