Validation and Comparison of Reference Genes for qPCR Normalization of Celery (Apium graveolens) at Different Development Stages

Abstract

A suitable reference gene is an important prerequisite for guarantying accurate and reliable results in qPCR analysis. Celery is one of the representative vegetable in Apiaceae and is widely cultivated and consumed in the world. However, no reports have been previously published concerning reference genes in celery. In this study, the expression stabilities of nine candidate reference genes in leaf blade and petiole at different development stages were evaluated using three statistics algorithms geNorm, NormFinder, and BestKeeper. Our results showed that TUB-B, TUB-A, and UBC were the most reference genes among all tested samples. GAPDH represented the maximum stability for most individual sample, while the UBQ displayed the minimum stability. To further validate the stability of reference genes, the expression pattern of AgAP2-2 was calculated by using the selected genes for normalization. In addition, the expression patterns of several development-related genes were studied using the selected reference gene. Our results will be beneficial for further studies on gene transcription in celery.

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

“…Primer amplification efficiency indicates the amplicon doubling rate of a specific primer pair during a PCR, which affects the accuracy of RT-qPCR data [4,40]. A good primer combination has an E value close to 100% [34,35]. In our study, the E values of the primers used to amplify the five candidate reference genes were between 94% and 98.5%.…”

“…In these studies, it is vital to choose appropriate reference genes for obtaining accurate and reliable results. A good primer combination has an E value close to 100% [34,35]. However, there has been no systematic and accurate validation of these genes under various experimental conditions.…”

“…Primer specificity was checked by melting curve and agarose gel electrophoresis analyses. Primers were only used in subsequent experiments if the condition 90% ≤ E ≤ 110% was met [34,35], and the mean squared error of the single data points fit to the standard curve (Error) value was below 0.2, as recommended by the Light-CyclerÒ 480 Software user manual. Primers were only used in subsequent experiments if the condition 90% ≤ E ≤ 110% was met [34,35], and the mean squared error of the single data points fit to the standard curve (Error) value was below 0.2, as recommended by the Light-CyclerÒ 480 Software user manual.…”

Identification of Reference Genes for qRT‐PCR Gene Expression Studies during Seed Development and under Abiotic Stresses in Cyamopsis tetragonoloba

“…Hence, a normalization method is required for obtaining reliable and unbiased quantitative gene expression profiling of a target gene. (Reddy et al, 2015), and Apium graveolens L. (Li et al, 2016), have shown that the expression of the HKGs is not as stable as it was generally assumed. The reliability of the quantitative results of qRT-PCR depends on the selected reference gene(s).…”

“…(Chandna et al, 2012), Arachis hypogea L. (Reddy et al, 2013), Vitis vinifera L. (Borges et al, 2014), Pennisetum glaucum (L.) R. Br. (Reddy et al, 2015), and Apium graveolens L. (Li et al, 2016), have shown that the expression of the HKGs is not as stable as it was generally assumed. It is risky to randomly choose any HKG to serve as a reference gene for qRT-PCR.…”