Reference Genes for RT-qPCR Analysis of Environmentally and Developmentally Regulated Gene Expression in Alfalfa

Abstract

Reverse transcription quantitative PCR (RT-qPCR) is a highly sensitive technique that has become the standard for the analysis of differences in gene expression in response to experimental treatments or among genetic sources. The accuracy of the RT-qPCR results can be significantly affected by uncontrolled sources of variation that can be accounted for normalization with so-called reference genes stably expressed under various conditions. In this study we assessed the stability of 21 reference gene candidates in crowns of two alfalfa cultivars (Apica and Evolution) exposed to various environmental conditions (cold, water stress and photoperiod) and from above ground biomass of the cultivar Orca sampled at three developmental stages (vegetative, full bloom and mature pods). Candidates were selected based on their previous identification in other plant species or their stable expression in a differential hybridization of alfalfa ESTs with cDNA from nonacclimated and cold-acclimated alfalfa. Genes encoding ubiquitin protein ligase 2a (UBL-2a), actin depolymerizing factor (ADF) and retention in endoplasmic reticulum 1 protein (Rer1) were the most stable across experimental conditions. Conversely β-actin (Act), α-tubulin (Tub) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) frequently used as "housekeeping genes" in gene expression studies showed poor stability. No more than two reference genes were required to normalize the gene expression data under each condition. Normalization of the expression of genes of interest with unstable reference genes led to observations that were conflicting with those made with validated reference genes and that were in some cases inconsistent with the current knowledge of the trait. The reference genes identified in this study are strong candidates for normalization of gene expression in cultivated alfalfa.

Melatonin Application Improves Salt Tolerance of Alfalfa (Medicago sativa L.) by Enhancing Antioxidant Capacity

“…The Na + and K + content was measured according to our previous work [62]. The shoot and root parts of alfalfa after different treatments were harvested and dried at 65 • C for 48 h. Approximately 50 mg of dry powder samples were sampled into 10 mL plastic tubes and 8 mL of deionized water was added.…”

Selection of reference genes for RT-qPCR analysis in the bark of Populus yunnanensis cuttings

“…Therefore, reference genes (internal control genes) are simultaneously applied to compensate these variations (Kumar et al, 2018;Park et al, 2015;Suzuki et al, 2000). Housekeeping genes such as 18S ribosomal RNA (18S rRNA), actin (ACT), tubulin (TUB), ubiquitin (UBQ), histone (HIS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are often used as traditional reference genes to normalize mRNA expression because of their constitutive expression or their involvement in maintenance of cell structure (Castonguay et al, 2015;Grbić et al, 2011;Khan et al, 2018;Li et al, 2018).…”

Identification of the regulatory networks and hub genes controlling alfalfa floral pigmentation variation using RNA-sequencing analysis

“…The primers were designed with the Primer premier 5.0 software and synthesized by TsingKe Biological Technology Co., Ltd. (Xi'an, China) (Table S1). Rer1 (JZ818481) was used as an internal standard [39]. The relative expression levels of genes were calculated using the 2 −ΔΔCt method [40].…”