Comprehensive Selection of Reference Genes for Gene Expression Normalization in Sugarcane by Real Time Quantitative RT-PCR

Abstract

The increasingly used real time quantitative reverse transcription-PCR (qRT-PCR) method for gene expression analysis requires one or several reference gene(s) acting as normalization factor(s). In order to facilitate gene expression studies in sugarcane (Saccharum officinarum), a non-model plant with limited genome information, the stability of 13 candidate reference genes was evaluated. The geNorm, NormFinder and deltaCt methods were used for selecting stably expressed internal controls across different tissues and under various experimental treatments. These results revealed that, among these 13 candidate reference genes, GAPDH, eEF-1a and eIF-4α were the most stable and suitable for use as normalization factors across all various experimental samples. In addition, APRT could be a candidate for examining the relationship between gene copy number and transcript levels in sugarcane tissue samples. According to the results evaluated by geNorm, combining CUL and eEF-1α in hormone treatment experiments; CAC and CUL in abiotic stress tests; GAPDH, eEF-1a and CUL in all treatment samples plus CAC, CUL, APRT and TIPS-41 in cultivar tissues as groups for normalization would lead to more accurate and reliable expression quantification in sugarcane. This is the first systematic validation of reference genes for quantification of transcript expression profiles in sugarcane. This study should provide useful information for selecting reference genes for more accurate quantification of gene expression in sugarcane and other plant species.

Selection of Reference Genes for qRT-PCR Analysis of Gene Expression in Stipa grandis during Environmental Stresses

“…Extensive studies on reference gene stability and selection have been reported in various plants for different tissues and development stages, and under diverse stresses [14–22]. Traditionally, reference genes such as 18S rRNA , GAPDH (glyceraldehyde-3-phosphate dehydrogenase), ACT (β or γ actin), TUB (α or β tubulin), EF1α (elongation factor 1α), and UBQ (poly-ubiquitin) have been used most frequently as internal control genes [13, 17]. Recently, taking advantage of a variety of databases comprising data from microarrays, expressed sequence tags, and transcriptomes, other stably expressed genes have been used as novel reference genes for qRT-PCR, such as CUL (Cullin), UBCP (ubiquitin carrier protein) [17], CAP (adenylyl cyclase-associated protein), SKIP1 (ASK-interacting protein 1) [22], F-BOX (F-box/kelch-repeat protein) [23], PP2A (protein phosphatase 2A) [24], SAMDC (s-adenosyl methionine decarboxylase) [12, 25], and SNF (sucrose non fermenting-1 protein kinase) [26].…”

Validation of reference genes for quantitative real-time PCR during latex regeneration in rubber tree

“…More than chance, the usage of the reported suitable reference genes in a different species or an altered condition leads to misleading results. Recently, more candidate reference genes are isolated and identified using gene expression profile data in many plants, including Saccharum (Ling et al, 2014), Elaeis guineensis (Chan et al, 2014), Tectona grandis L.f. (Galeano et al, Gene xxx (2015) xxx-xxx Abbreviations: qRT-PCR, Quantitative real-time PCR; Ct, threshold cycle (previously); Cq, quantification cycle; HKGs, Housekeeping genes; SD, standard deviation; CV, coefficient of variation; 18S, 18s ribosomal RNA; ACT7a, Actin (ACTIN7); ACT7b, Actin (ACTIN7); ADF, Actin depolymerizing factor; ADF4, Actin depolymerizing factor 4; eIF1Aa, Eukaryotic translation initiation factor 1A; eIF1Ab, Eukaryotic translation initiation factor 1A; eIF2, Eukaryotic translation initiation factor; eIF3, Eukaryotic translation initiation factor; FP, F-box family protein; PTP, Trosine phosphatase; RH2a, DEAD box RNA helicase,RH2; RH2b, DEAD box RNA helicase,RH2; ROC3, Cytosolic cyclophilin (ROC3); UBC1, Ubiquitin-protein ligase; UBC2a, Ubiquitin-protein ligase (ATUBC2); UBC2b, Ubiquitin-protein ligase (ATUBC2); UBC3, Ubiquitin-protein ligase; UBC4, Ubiquitin-protein ligase; YLS8, Mitosis protein YLS8.…”

Identification of smut-responsive genes in sugarcane using cDNA-SRAP

“…Three differentially expressed genes of interest, topoisomerase gene (EU048780), ethylene insensitive gene (EU048778), and tetraspanin gene (EU048770), were selected for expression profile analysis. Primers specific to the sugarcane 25S rRNA gene were used for normalization of the reactions (Ling et al, 2014), and real-time qPCR primers were designed using Primer premier 5.0 (Table 2).…”