Validation of Suitable Reference Genes for Assessing Gene Expression of MicroRNAs in Lonicera japonica

Abstract

MicroRNAs (miRNAs), which play crucial regulatory roles in plant secondary metabolism and responses to the environment, could be developed as promising biomarkers for different varieties and production areas of herbal medicines. However, limited information is available for miRNAs from Lonicera japonica, which is widely used in East Asian countries owing to various pharmaceutically active secondary metabolites. Selection of suitable reference genes for quantification of target miRNA expression through quantitative real-time (qRT)-PCR is important for elucidating the molecular mechanisms of secondary metabolic regulation in different tissues and varieties of L. japonica. For precise normalization of gene expression data in L. japonica, 16 candidate miRNAs were examined in three tissues, as well as 21 cultivated varieties collected from 16 production areas, using GeNorm, NormFinder, and RefFinder algorithms. Our results revealed combination of u534122 and u3868172 as the best reference genes across all samples. Their specificity was confirmed by detecting the cycling threshold (Ct) value ranges in different varieties of L. japonica collected from diverse production areas, suggesting the use of these two reference miRNAs is sufficient for accurate transcript normalization with different tissues, varieties, and production areas. To our knowledge, this is the first report on validation of reference miRNAs in honeysuckle (Lonicera spp.). Restuls from this study can further facilitate discovery of functional regulatory miRNAs in different varieties of L. japonica.

Regulation of fatty acid and flavonoid biosynthesis by miRNAs in Lonicera japonica

“…A combination of the genes, U534122 and U3868172, was used as the internal control in the detection of miRNA expression by qRT-PCR. 2 The specic forward primers for the selected miRNAs (ESI Table 3 †) were obtained from Sangon Biotech (Shanghai, China).…”

“…1 This important herb is used to treat various diseases, such as H1N1 inuenza, severe acute respiratory syndromes, pancreatic cancer, and hand-foot-and-mouth disease. 2 A new type of anthocyanin-rich L. japonica tea, with its red colour, avour, and taste has become popular in the market. It is prepared from the buds of L. japonica Thunb.…”

Selection and validation of suitable reference genes for qPCR gene expression analysis in goats and sheep under Peste des petits ruminants virus (PPRV), lineage IV infection

“…geNorm also identifies the number of reference genes required for the normalization of a particular experiment 48 . The NormFinder allows comparison of intra- and inter-group variation and calculates expression stability value (M) 49 , 50 . BestKeeper uses repeated pairwise correlation analysis to determine the optimal reference genes 51 .…”

Selection and Evaluation of Candidate Reference Genes for Quantitative Real-Time PCR in Aboveground Tissues and Drought Conditions in Rhododendron Delavayi

“…One of the requisites for genomics and transcriptomics is qRT-PCR which measures changes in gene expression ( Phillips et al, 2009 ; Köhsler et al, 2020 ). The prominence of qRT-PCR is due to its precision, accuracy, convenience, speed, and sensitivity ( Wang et al, 2016 ). However, qRT-PCR requires a housekeeping/reference gene to act as an internal control to normalize the expression level of target genes ( Bustin et al, 2009 ; Gao et al, 2017 ).…”