Selection of reliable reference genes for quantitative real-time polymerase chain reaction studies in maize grains

Key message

The stability of candidate reference genes was evaluated in maize landrace varieties and during multiple grain developmental stages to evaluate the expression of carotenoid-related genes by RT-qPCR for application to maize biofortification.

Abstract

Vitamin A deficiency affects millions of children worldwide; therefore, increasing the content of vitamin A precursors in maize grains is of interest. The study of the expression of genes involved in the carotenoid biosynthetic pathway in maize grains has provided useful information for metabolic engineering approaches. However, reliable results using real-time quantitative polymerase chain reaction (RT-qPCR) experiments are dependent on the use of the appropriate reference genes. In this study, we utilized geNorm and NormFinder softwares to identify the most stably expressed candidate reference genes in samples from seven stages of grain development and from eight landrace varieties. The results of the analysis performed using geNorm indicated that tubulin (TUB) and actin (ACT) were the most suitable reference genes among all experimental conditions, while glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) showed the least stability. The same result was obtained with the NormFinder software. The minimum number of genes required in each experimental condition to normalize the gene expression data was also determined by geNorm. The expression of phytoene synthase gene (PSY1), the first enzyme in the carotenoid biosynthetic pathway, was overestimated when the least stable candidate gene (GAPDH) was used as the internal control instead of the most stable gene pair (ACT + TUB), thus highlighting the importance of validating reference genes before conducting a RT-qPCR experiment to obtain accurate results. This study is the first survey of the stability of genes for use as reference genes to normalize RT-qPCR data from maize landraces during multiple stages of grain development.     

Validation of reliability for reference genes under various abiotic stresses in tea plant

Reference genes are frequently used as a normalization standard to obtain reliable data during quantitative real-time polymerase chain reaction (qRT-PCR). However, recent studies showed that most traditional reference genes were not stable under different treatments or environmental stresses, which may lead to misinterpret expression of the target genes. In this study, 7 candidate reference genes in tea plant (Camellia sinensis (L.) Kuntze cv. Yingshuang) were selected and their expression stability under different abiotic stresses was analyzed using geNorm, NormFinder, and BestKeeper methods. Our results suggest that TUA1 (alpha-1 tubulin) has the most stable expression under damage stresses according to 3 methods of analysis. For drought stresses, 18S rRNA, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were the most stable genes. For cold, Al, and NaCl stresses, GAPDH and TUA1 may be the alternative options. Our results may provide an insight for identification of the optimal reference genes for tea plants under various treatments.     

Validation of reference genes in the feline endometrium

The aim of the study was to find the most stable reference genes from: ACTB, GAPDH, RPL30, CYC, RPL17, RPS7 and YWHAZ in the feline endometrium. Three free software packages, geNorm, NormFinder and BestKeeper were used. In geNorm analysis, the most stable gene was RPS7 (at a primer concentration 1000 nM) or YWHAZ (500 and 250 nM). According to NormFinder and BestKeeper, ACTB (at all examined primer concentrations) followed by RPS7 and CYC were the most stable genes. Based on geNorm results at least two genes from among RPS7, RPL30, ACTB or YWHAZ should be chosen for Real Time-PCR result normalization.     

斑地锦RT-qPCR内参基因的筛选

实时荧光定量PCR(RT-qPCR)的前提条件之一是具有合适的内参基因。为筛选斑地锦(Euphorbia maculata)合适的RT-qPCR内参基因,该文利用同源克隆法克隆斑地锦GAPDH、EF-1α、act、UBQ、TUB-α、eIF-4A、CYP等基因片段,RT-qPCR检测7个候选内参基因在斑地锦不同生长期根、茎、叶和果实中的表达情况,并用geNorm、NormFinder和BestKeeper等生物学软件对各候选基因表达稳定性进行评价。结果表明:(1)克隆的GAPDH、EF-1α、act、UBQ、TUB-α、eIF-4A、CYP基因片段为729、808、753、422、233、656、313 bp,分别编码242、269、250、140、77、218、103个氨基酸,与其他植物相应氨基酸序列的最高同源性均在85%以上。(2)综合3个分析软件分析内参基因表达稳定性得出,表达稳定性排名为UBQ>EF-1α>TUB-α>eIF-4A>GAPDH>CYP>act。因此,可以选取UBQ作为斑地锦RT-qPCR分析的内参基因,用于不同生长期基因组织特异性表达研究。