Expression Stabilities of Ten Candidate Reference Genes for RT-qPCR in Zanthoxylum bungeanum Maxim

Abstract

Real-time reverse transcription quantitative PCR has become a common method for studying gene expression, however, the optimal selection of stable reference genes is a prerequisite for obtaining accurate quantification of transcript abundance. Suitable reference genes for RT-qPCR have not yet been identified for Chinese prickly ash (Zanthoxylum bungeanum Maxim.). Chinese prickly ash is the source of an important food seasoning in China. In recent years, Chinese prickly ash has also been developed as a medicinal plant. The expression stabilities of ten genes (18S, 28S, EF, UBA, UBQ, TIF, NTB, TUA, RPS, and TIF5A) were evaluated in roots, stems, leaves, flowers and fruits at five developmental stages and also under stress from cold, drought, and salt. To do this we used three different statistical algorithms: geNorm, NormFinder and BestKeeper. Among the genes investigated, UBA and UBQ were found to be most stable for the different cultivars and different tissues examined, UBQ and TIF for fruit developmental stage. Meanwhile, EF and TUA were most stable under cold treatment, EF and UBQ under drought treatment and NTB and RPS under salt treatment. UBA and UBQ for all samples evaluated were most stably expressed, but 18S, TUA and RPS were found to be generally unreliable as reference genes. Our results provide a basis for the future selection of reference genes for biological research with Chinese prickly ash, under a variety of conditions.

RNA‐seq‐based selection of reference genes for RT‐qPCR analysis of pitaya

“…The reliability of RT-qPCR data is affected by the reference gene and sample preparation [4]. Their expression shows large variations in different tissues, developmental stages, and under different stresses [8][9][10][11]. However, an increasing number of studies have shown that none of the common reference genes meet this criterion for all of the test conditions.…”

Identification of microRNA-target interactions for antioxidant defense response under drought stress by high-throughput sequencing in Zanthoxylum bungeanum Maxim

“…The qRT-PCR assays were carried out on a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The reaction system was of 10 μl, containing 5 μl of 2× SYBR Premix Ex Taq II (TaKaRa), 1 μl of cDNA, 1 μl of each of the upstream and downstream primers and 2 μl of ddH 2 O. ZbUBQ and Zbα-EF were used for the reference genes to correct the RT-qPCR data (Fei et al, 2018). The RT-qPCT reaction system of miRNAs is identical to mRNA, with a reaction system of 10 μL, containing 5 μL of 2× SYBR Premix Ex Taq II (TaKaRa, Beijing, China), 1 μL of cDNA, 1 μL of each of the forward and universal reverse primer and 2 μL of ddH 2 O. U6 was used for the correction of relative expression levels of miRNAs (Zhang et al, 2018).…”

De novo transcriptome assembly for the five major organs of Zanthoxylum armatum and the identification of genes involved in terpenoid compound and fatty acid metabolism

“…qRT-PCR was performed in a Bio-Rad CFX96™ system (Bio-Rad, America) with SYBR Premix Ex Taq™ II (TaKaRa, Japan). ZaUBQ was selected as an internal control according to a previous study [2]. The 2 -ΔΔCt method was used to calculate the relative expression levels of the genes in different samples [63].…”

“…Zanthoxylum armatum (Rutaceae), commonly known as green Sichuan pepper, is one of the most economically important trees in Sichuan Province and is widely distributed in most parts of southwest China and some parts of southeast Asia [1]. The production of Z. armatum is currently a billions of dollars commercially, and this species has a long history of cultivation in China because it is one of the eight main spices used in Chinese cuisine and is an essential ingredient in Sichuan hot-pot, with a special numbing taste [2]. However, the underlying regulatory mechanism of the genes associated with the numbing taste remains poorly understood.…”