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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 (C t ) 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.    Conclusion   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.

chinese medicine

Selection and validation of reference genes for normalization of quantitative real-time reverse transcription PCR analysis in Poria cocos (Schw.) Wolf (Fuling)