Validation of reference genes for gene expression analysis in Valsa mali var. mali using real-time quantitative PCR

Valsa mali var. mali (Vmm), is the predominant species of apple valsa canker in China. Modern analysis of genes involved in virulence or pathogenicity usually implicate gene expression analysis most often performed using real-time quantitative polymerase chain reaction (RT-qPCR). However, for relative gene expression analysis pertinent reference genes have to be validated before using them as internal reference. This has not been reported for Vmm, so far. Therefore, eight commonly used housekeeping genes (ACT, CYP, EF1-α, G6PDH, GAPDH, L13, TUB, and UBQ) were cloned and evaluated for their expression stability by geNorm and NormFinder. Overall, all of the candidate reference genes were found to be suitable for gene expression analysis. After analysis of 10 samples from different strains and abiotic stress treatments, G6PDH appeared to be the most suitable reference gene, whereas GAPDH was the least suitable. Moreover, taking G6PDH combined with L13 or CYP as reference genes, improved the reliability of RT-qPCR significantly. The influence of the reference system on expression data was demonstrated by analyzing Vmmpg-1 encoding an endo-polygalacturonase gene. Pectinases are considered key pathogenicity factors for this fungus. In order to better understand the role of pectinases in pathogenicity of Vmm, RT-qPCR was used for expression analysis. Our results may provide a guideline for future studies on gene expression of V. mali var. mali by using RT-qPCR.     

Selection of candidate reference genes for real-time PCR studies in lettuce under abiotic stresses

The process of selection and validation of reference genes is the first step in studies of gene expression by real-time quantitative polymerase chain reaction (RT-qPCR). The genome of lettuce, the most popular leaf vegetable cultivated worldwide, has recently been sequenced; therefore, suitable reference genes for reliable results in RT-qPCR analyses are required. In the present study, 17 candidate reference genes were selected, and their expression stability in lettuce leaves under drought, salt, heavy metal, and UV-C irradiation conditions and under the application of abscisic acid (ABA) was evaluated using geNorm and NormFinder software. The candidate reference genes included protein-coding traditional and novel reference genes and microRNAs (miRNAs). The results indicate that the expression stability is dependent on the experimental conditions. The novel protein-coding reference genes were more suitable than the traditional reference genes under drought, UV-C irradiation, and heavy metal conditions and under the application of ABA. Only under salinity conditions were the traditional protein-coding reference genes more stable than the novel genes. In addition, the miRNAs, mainly MIR169, MIR171/170 and MIR172, were stably expressed under the abiotic stresses evaluated, representing a suitable alternative approach for gene expression data normalization. The expression of phenylalanine ammonia lyase (PAL) and 4-hydroxyphenylpyruvate dioxygenase (HPPD) was used to further confirm the validated protein-coding reference genes, and the expression of MIR172 and MIR398 was used to confirm the validated miRNA genes, showing that the use of an inappropriate reference gene induces erroneous results. This work is the first survey of the stability of reference genes in lettuce and provides guidelines to obtain more accurate RT-qPCR results in lettuce studies.     

Screening of valid reference genes for real-time RT-PCR data normalization in Hevea brasiliensis and expression validation of a sucrose transporter gene HbSUT3

Real-time RT-PCR (RT-qPCR) is a sensitive and precise method of quantifying gene expression, however, suitable reference genes are required. Here, a systematic reference gene screening was performed by RT-qPCR on 22 candidate genes in Hevea brasiliensis. Two ubiquitin-protein ligases (UBC2a and UBC4) were the most stable when all samples were analyzed together. A mitosis protein (YLS8) and a eukaryotic translation initiation factor (eIF1Aa) were the most stable in response to tapping. UBC2b and UBC1 were the most stable among different genotypes. UBC2b and a DEAD box RNA helicase (RH2b) were the most stable across individual trees. YLS8 and RH8 were most stably expressed in hormone-treated samples. Expression of the candidate reference genes varied significantly across different tissues, and at least four genes (RH2b, RH8, UBC2a and eIF2) were needed for expression normalization. In addition, examination of relative expression of a sucrose transporter HbSUT3 in different RNA samples demonstrated the importance of additional reference genes to ensure accurate quantitative expression analysis. Overall, our work serves as a guide for selection of reference genes in RT-qPCR gene expression studies in H. brasiliensis.