Open AccessSelection and validation of reference genes for normalization of qRT-PCR data to study the cannabinoid pathway genes in industrial hemp
Author(s) -
Michihito Deguchi,
Shobha Potlakayala,
Zachary Spuhler,
Hannah George,
Vijay Sheri,
Ruba Agili,
Aayushi Patel,
Sairam Rudrabhatla
Publication year - 2021
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0260660
Subject(s) - reference genes , gene , biology , computational biology , gene expression , normalization (sociology) , genetics , candidate gene , gene expression profiling , bioinformatics , sociology , anthropology
There has been significant interest in researching the pharmaceutical applications of Industrial hemp since its legalization three years ago. The crop is mostly dioecious and known for its production of phytocannabinoids, flavonoids, and terpenes. Although many scientific reports have showed gene expression analysis of hemp through OMICs approaches, unreliable reference genes for normalization of qRT-PCR data make it difficult to validate the OMICs data. Four software packages: geNorm, NormFinder, BestKeeper, and RefFinder were used to evaluate the differential gene expression patterns of 13 candidate reference genes under osmotic, heavy metal, hormonal, and UV stresses. EF-1α ranked as the most stable reference gene across all stresses, TUB was the most stable under osmotic stress, and TATA was the most stable under both heavy metal stress and hormonal stimuli. The expression patterns of two cannabinoid pathway genes, AAE1 and CBDAS , were used to validate the reliability of the selected reference genes. This work provides useful information for gene expression characterization in hemp and future research in the synthesis, transport, and accumulation of secondary metabolites.