Validation of Zebrafish （Danio redo） Reference Genes for Quantitative Real-time RT-PCR Normalization

The normalization of quantitative real time RT-PCR （qRT-PCR） is important to obtain accurate gene expression data. The most common method for qRT-PCR normalization is to use reference, or housekeeping genes. However, there is emerging evidence that even reference genes can be regulated under different conditions, qRT-PCR has only recently been used in terms of zebrafish gene expression studies and there is no validated set of reference genes. This study characterizes the expression of nine possible reference genes during zebrafish embryonic development and in a zebrafish tissue panel. All nine reference genes exhibited variable expression. The fl-actin, EFlot and Rpll3ot genes comprise a validated reference gene panel for zebrafish developmental time course studies, and the EF1 or, Rpll3α and 18S rRNA genes are more suitable as a reference gene panel for zebrafish tissue analysis. Importantly, the zebrafish GAPDH gene appears unsuitable as reference gene for both types of studies.     

Identification of suitable reference genes for quantitative real‐time PCR normalization in blotched snakehead Channa maculata

A systematic study was conducted to identify reliable reference genes for normalization of gene expression analysis in the blotched snakehead Channa maculata under normal physiological conditions. Firstly, the partial complementary (c)DNA of nine candidate reference genes (actb, tmem104, ube2l3, ef1α, churc1, tmem256, rpl13a, sep15 and g6pd) were cloned from C. maculata. The expression levels of these genes were then assessed in embryos of different developmental stages and various tissue types of adult fish using quantitative real‐time (qrt‐)PCR. RefFinder algorithm was used to evaluate the expression stability of these genes based on their cycle‐threshold (C_t) values in the qrt‐PCR analysis. Results showed that there was no single best reference gene for all stages of embryos and adult tissues tested. Furthermore, it was found that, among the nine candidate genes tested, actb and tmem104 were the most stable reference genes across adult tissue types, while sep15 and tmem256 were the most stable ones across developmental stages of embryos. These stable reference genes are recommended for normalization of gene expression analysis in C. maculata.     

Ontogeny of odorant receptor gene expression in zebrafish,Danio rerio

We cloned three putative odorant receptor (OR) genes from the zebrafish to use as in situ hybridization probes to follow the temporal patterns of neurons expressing OR genes through a developmental progression from embryo (12 h postfertilization) to adult. The identification of these genes is supported by sequence homology to previously reported ORs and by the morphology and location of labeled cells in in situ hybridization experiments. Cells expressing OR mRNA were first observed in the olfactory placodes between 31 and 38 h after fertilization (fish reared at 26°C). Initially, only single cells were observed to hybridize the probe; the number of labeled cells increased throughout the remainder of embryogenesis and through postembryonic growth and morphogenesis of the olfactory organ. At all ages, the positively hybridizing cells were scattered throughout the olfactory epithelium but not in the nonsensory epithelium of the olfactory organ. © 1996 John Wiley & Sons, Inc.     

Validation of reference genes for RT-qPCR analysis of CYP4T expression in crucian carp

Reference genes are commonly used for normalization of target gene expression during RT-qPCR analysis. However, no housekeeping genes or reference genes have been identified to be stable across different tissue types or under different experimental conditions. To identify the most suitable reference genes for RT-qPCR analysis of target gene expression in the hepatopancreas of crucian carp (Carassius auratus) under various conditions (sex, age, water temperature, and drug treatments), seven reference genes, including beta actin (ACTB), beta-2 microglobulin (B2M), embryonic elongation factor-1 alpha (EEF1A), glyceraldehyde phosphate dehydrogenase (GAPDH), alpha tubulin (TUBA), ribosomal protein l8 (RPL8) and glucose-6-phosphate dehydrogenase (G6PDH), were evaluated in this study. The stability and ranking of gene expression were analyzed using three different statistical programs: GeNorm, Normfinder and Bestkeeper. The expression errors associated with selection of the genes were assessed by the relative quantity of CYP4T. The results indicated that all the seven genes exhibited variability under the experimental conditions of this research, and the combination of ACTB/TUBA/EEF1A or of ACTB/EEF1A was the best candidate that raised the accuracy of quantitative analysis of gene expression. The findings highlighted the importance of validation of housekeeping genes for research on gene expression under different conditions of experiment and species.     

Identification and validation of reference genes for RT‐qPCR analysis in banana (Musa spp.) under Fusarium wilt resistance induction conditions

Banana (Musa spp.) is severely damaged by Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc). Biocontrol by inducing systemic resistance has been considered as one of the most important strategies to improve plant health. Very few studies have investigated appropriate reference gene selection for RT‐qPCR (quantitative real‐time polymerase chain reaction) analysis suitable for conditions of systemic activated resistance. In this study, we assessed over a time‐course the expression of seven candidate reference genes (EF1, TUB, ACT1, ACT2, L2, RPS2 and RAN) for Cavendish cultivar Brazilian (Musa spp. AAA) and dwarf banana cultivar Guangfen No. 1 (Musa spp. ABB) that were inoculated by Bacillus subtilis strain TR21 and Foc. We choose these plants because they are commonly planted in Southern China. Expression stability of the candidate genes was evaluated using various software packages (GeNorm, NormFinder and BestKeeper). L2 and TUB genes displayed maximum stability in Guangfen No. 1. In Brazilian, ACT1 and TUB were the most stable genes. To further validate the suitability of the reference genes identified in this study, the expression of pathogenesis‐related 1 (PR1) gene under TR21 and Foc strains Foc004/Foc009 treatments was also studied. Identified reference genes in this work that are most suitable for normalizing gene expression data in banana under Fusarium wilt resistance induction conditions will contribute to the understanding of disease resistance mechanisms induced by biocontrol strains in banana.     

Validation of Zebrafish (Danio rerio) Reference Genes for Quantitative Real-time RT-PCR Normalization

The normalization of quantitative real time RT-PCR (qRT-PCR) is important to obtain accurate gene expression data. The most common method for qRT-PCR normalization is to use reference, or house- keeping genes. However, there is emerging evidence that even reference genes can be regulated under different conditions, qRT-PCR has only recently been used in terms of zebrafish gene expression studies and there is no validated set of reference genes. This study characterizes the expression of nine possible reference genes during zebrafish embryonic development and in a zebrafish tissue panel. All nine reference genes exhibited variable expression. The β-actin, EF1α and Rp113α genes comprise a validated reference gene panel for zebrafish developmental time course studies, and the EF1α, Rp113α and 18S rRNA genes are more suitable as a reference gene panel for zebrafish tissue analysis. Importantly, the zebrafish GAPDH gene appears unsuitable as reference gene for both types of studies.     

PlantRGS: a web server for the identification of most suitable candidate reference genes for quantitative gene expression studies in plants

Normalization of quantitative gene expression data with a suitable reference gene is essential for accurate and reliable results. However, the availability and choice of most suitable reference gene(s) showing uniform expression across all the experimental conditions remain a drawback. We have developed a web server, PlantRGS (http://www.nipgr.res.in/PlantRGS), for the identification of most suitable candidate reference gene(s) at the whole-genome level using microarray data for quantitative gene expression studies in plants. Microarray data from more than 11 000 tissue samples for nine plant species have been included in the PlantRGS for meta-analysis. The web server provides a user-friendly graphical user interface-based analysis tool for the identification of most suitable reference genes in the selected plant species under user-defined experimental conditions. Various parameter options and output formats will help users to investigate desired number of most suitable reference genes with wide range of expression levels. Validation of results revealed that novel reference genes identified by the PlantRGS outperforms the traditionally used reference genes in terms of expression stability. We anticipate that the PlantRGS will provide a platform for the identification of most suitable reference gene(s) under given experimental conditions and facilitate quantitative gene expression studies in plants.     

Identification of valid reference genes for the normalization of RT qPCR gene expression data in human brain tissue

Background

Transfection of cells with gene-specific, single-stranded oligonucleotides can induce the targeted exchange of one or two nucleotides in the targeted gene. To characterize the features of the DNA-repair mechanisms involved, we examined the maximal distance for the simultaneous exchange of two nucleotides by a single-stranded oligonucleotide. The chosen experimental system was the correction of a hprt-point mutation in a hamster cell line, the generation of an additional nucleotide exchange at a variable distance from the first exchange position and the investigation of the rate of simultaneous nucleotide exchanges.

Results

The smaller the distance between the two exchange positions, the higher was the probability of a simultaneous exchange. The detected simultaneous nucleotide exchanges were found to cluster in a region of about fourteen nucleotides upstream and downstream from the first exchange position.

Conclusion

We suggest that the mechanism involved in the repair of the targeted DNA strand utilizes only a short sequence of the single-stranded oligonucleotide, which may be physically incorporated into the DNA or be used as a matrix for a repair process.     

Genome‐wide expression quantitative trait locus studies facilitate isolation of causal genes controlling panicle structure

The morphology of rice (Oryza sativa L.) panicles is an important determinant of grain yield, and elucidation of the genetic control of panicle structure is very important for fulfilling the demand for high yield in breeding programs. In a quantitative trait locus (QTL) study using 82 backcross inbred lines (BILs) derived from Koshihikari and Habataki, 68 QTLs for 25 panicle morphological traits were identified. Gene expression profiling from inflorescence meristems of BILs was obtained. A combination of phenotypic QTL (pQTL) and expression QTL (eQTL) analysis revealed co‐localization between pQTLs and eQTLs, consistent with significant correlations between phenotypic traits and gene expression levels. By combining pQTL and eQTL data, two genes were identified as controlling panicle structure: OsMADS18 modulates the average length of the primary rachis and OsFTL1 has pleiotropic effects on the total number of secondary rachides, number of grains per panicle, plant height and the length of flag leaves. Phenotypes were confirmed in RNA interference knocked‐down plants and overexpressor lines. The combination of pQTL and eQTL analysis could facilitate identification of genes involved in rice panicle formation.     

Genome‐wide characterization and expression analysis of genetic variants in sweet orange

Heterozyosity is an important feature of many plant genomes, and is related to heterosis. Sweet orange, a highly heterozygous species, is thought to have originated from an inter‐species hybrid between pummelo and mandarin. To investigate the heterozygosity of the sweet orange genome and examine how this heterozygosity affects gene expression, we characterized the genome of Valencia orange for single nucleotide variations (SNVs), small insertions and deletions (InDels) and structural variations (SVs), and determined their functional effects on protein‐coding genes and non‐coding sequences. Almost half of the genes containing large‐effect SNVs and InDels were expressed in a tissue‐specific manner. We identified 3542 large SVs (>50 bp), including deletions, insertions and inversions. Most of the 296 genes located in large‐deletion regions showed low expression levels. RNA‐Seq reads and DNA sequencing reads revealed that the alleles of 1062 genes were differentially expressed. In addition, we detected approximately 42 Mb of contigs that were not found in the reference genome of a haploid sweet orange by de novo assembly of unmapped reads, and annotated 134 protein‐coding genes within these contigs. We discuss how this heterozygosity affects the quality of genome assembly. This study advances our understanding of the genome architecture of sweet orange, and provides a global view of gene expression at heterozygous loci.     

Effect of chilling on sox2, sox3 and sox19a gene expression in zebrafish (Danio rerio) embryos

Zebrafish embryos have not been cryopreserved due to their structural limitations. Although embryo survival rates have been used as the measured outcome for most of the cryopreservation protocols studied, there are very limited data available at the molecular level. This study focused on the effect of chilling and subsequent warming on gene expression of sox2, sox3 and sox19a which play vital roles in the development of zebrafish embryos. A quantitative RT-PCR approach was used to investigate gene expression following chilling at 0 °C for up to 180 min. The effect on gene expression was also studied during a 180 min warming period after chilling for 30 or 60 min. There were significant decreases in sox2 (up to 4-fold) and sox3 (up to 3-fold) expressions following chilling. Significant increases in gene expressions of sox2 (up to 2-fold), sox3 (up to 33-fold) and sox19a (up to 25-fold) were observed during warming in the embryos that had been chilled for 30 min. Similarly, significant increases were observed in sox2 (up to 3-fold) and sox3 (up to 2-fold) during warming in embryos that had been chilled for 60 min. These increases may be explained by compensation for the suppression observed during chilling and/or to activate repair mechanisms or maintain homeostasis.