Selection and evaluation of housekeeping genes as endogenous controls for quantification of mRNA transcripts in Theileria parva using quantitative real-time polymerase chain reaction (qPCR)

The reliability of any quantitative real-time polymerase chain reaction (qPCR) experiment can be seriously compromised by variations between samples as well as between PCR runs. This usually result from errors in sample quantification, especially with samples that are obtained from different individuals and tissues and have been collected at various time intervals. Errors also arise from differences in qPCR efficiency between assays performed simultaneously to target multiple genes on the same plate. Consequently, the derived quantitative data for the target genes become distorted. To avoid this grievous error, an endogenous control, with relatively constant transcription levels in the target individual or tissue, is included in the qPCR assay to normalize target gene expression levels in the analysis. Several housekeeping genes (HKGs) have been used as endogenous controls in quantification studies of mRNA transcripts; however, there is no record in the literature of the evaluation of these genes for the tick-borne protozoan parasite, Theileria parva. Importantly, the expression of these genes should be invariable between different T. parva stocks, ideally under different experimental conditions, to gain extensive application in gene expression studies of this parasite. Thus, the expression of several widely used HKGs was evaluated in this study, including the genes encoding β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 28S rRNA, cytochrome b and fructose-2.6-biphosphate aldolase (F6P) proteins. The qPCR analysis revealed that the expression of genes encoding cytochrome b, F6P and GAPDH varied considerably between the two T. parva stocks investigated, the cattlederived T. parva Muguga and the buffalo-derived T. parva 7014. 28S rRNA and β-actin gene expression was the most stable; thus, these genes were considered suitable candidates to be used as endogenous control genes for mRNA quantification studies in T. parva.This work was funded by the Genomics Research Institute of the University of Pretoria, (http://www.up.ac.za/the-genomics-research- institute/home) to KPS-M.http://www.plosone.orgam2018Veterinary Tropical Disease

Validation of expression profiles of differentially expressed transcripts identified in cattle-derived and buffalo-derived Theileria parva isolates by RNA sequencing

The intracellular apicomplexan protozoan parasite Theileria parva is a causative agent of cattle theileriosis which manifests in two disease syndromes, namely East Coast fever (ECF) and Corridor disease. Although ECF was eradicated from South Africa, cattle theileriosis still persists in the form of Corridor disease. Moreover, it is not known if the T. parva parasites present in buffalo in South Africa could cause ECF if they were to become established in cattle. This has made it essential to identify genetic differences that would allow successful discrimination of cattle-derived (causative agents of ECF) and buffalo-derived (causative agents of Corridor disease) T. parva parasites. Consequently, Next Generation Sequencing (NGS) was utilized to analyse T. parva transcriptomes from two isolates representing cattle-derived and buffalo-derived parasites, in order to identify gene expression profiles that may characterize cattle-derived and buffalo-derived T. parva isolates. However, RNA-sequencing (RNA-seq) experiments can be influenced by variability caused by technical effects including multiple template preparation stages, diverse sequencing chemistries and complex data processing of NGS experiments; it is thus crucial that data from these experiments is validated using other technologies. Thus, the aim of this study was to use quantitative real-time polymerase chain reaction (qPCR) for validation of differentially expressed genes (DEGs) identified from the RNA-seq study using NGS. Three groups of genes representing different expression profiles, including: 1. constitutively expressed genes; 2. up- and down-regulated genes and 3. genes exclusively expressed in one isolate or the other, were selected for validation. Prior to validation of expression profiles for the selected set of genes using qPCR, endogenous control genes had to be selected in order to normalize qPCR gene expression data. Since there is no information available on the evaluation of the expression stability of these genes in T. parva isolates, the expression stability of five candidate reference genes, β-actin, glyceraldehyde-3-phosphate dehydrogenase, 28S rRNA, cytochrome b and fructose bisphosphatase aldolase (F6P), was evaluated for identification of reliable reference genes. The outcome of the stability rankings for each gene varied according to the program showing that the criteria for stability ranking differ from program to program. It is for this reason that the RefFinder tool, used in this study, integrates the different programs and gives a recommended comprehensive ranking. Therefore, based on this comprehensive analysis between the two T. parva isolates investigated, 28S rRNA and β-actin genes were selected as most suitable reference genes for this study. Intra- and inter-assay variation analysis of the selected reference genes showed that there was no significant variation in the expression of these genes between the two T. parva isolates with the p values being less than 0.05 and the coefficient of variation percentage being low (<2) for all the genes tested. Thus, we propose that genes coding for 28S rRNA and β-actin proteins be employed as endogenous control genes in studies that involve gene expression analysis of T. parva. Validation of expression profiles from RNA-seq data obtained using NGS was performed using qPCR. In this study, the comparative CT method for qPCR data analysis was employed to analyse the expression profiles of selected genes. The use of this method requires initial validation by ensuring that the target genes have approximately the same amplification efficiency as the endogenous control genes. Therefore, the amplification efficiencies of target genes and endogenous control genes were evaluated by constructing validation plots from standard curves generated from selected constitutively expressed and differentially expressed genes, in comparison to the standard curves of the two endogenous control genes. Initially, cDNA was prepared from total RNA isolated from bovine and buffalo lymphoblastoid cell cultures infected with Theileria parva (Muguga) and Theileria parva (7014), respectively, previously used for RNA-seq by NGS. The quantity of the parasite cDNA from the two isolates was interpolated from the standard curve and standardized to a concentration of 36.03 ng/μl, to eliminate concentration bias in downstream gene expression analysis. This study passed the comparative CT method validation experiment since the absolute slopes of ΔCT vs. Log input cDNA for the selected target genes were all less than 0.1 as required. Twenty DEGs, constituting up- and down-regulated genes and genes exclusively expressed in one isolate or the other, and 10 stably expressed (constitutive) genes were selected for validation of expression profiles from RNA-seq data obtained using NGS. Discrepancies between RNA-seq and qPCR analyses were observed from all three groups of target genes but mostly in the constitutively expressed group of genes; in this group only 40% of the qPCR results corroborated with RNA-seq findings while 60% demonstrated variations in expression with four genes down-regulated and one up-regulated in T. parva 7014 relative to T. parva Muguga. Since most of the disagreements in the two datasets were down-regulated expression, this finding suggests that RNA-seq was more sensitive in detecting low abundant RNA transcripts.Dissertation (MSc)--University of Pretoria, 2017.Veterinary Tropical DiseasesMScUnrestricte

Bacillus for Plant Growth Promotion and Stress Resilience: What Have We Learned?

The rhizosphere is a thin film of soil that surrounds plant roots and the primary location of nutrient uptake, and is where important physiological, chemical, and biological activities are occurring. Many microbes invade the rhizosphere and have the capacity to promote plant growth and health. Bacillus spp. is the most prominent plant growth promoting rhizobacteria due to its ability to form long-lived, stress-tolerant spores. Bacillus-plant interactions are driven by chemical languages constructed by a wide spectrum of metabolites and lead to enhanced plant growth and defenses. Thus, this review is a synthesis and a critical assessment of the current literature on the application of Bacillus spp. in agriculture, highlighting gaps that remain to be explored to improve and expand on the Bacillus-based biostimulants. Furthermore, we suggest that omics sciences, with a focus on metabolomics, offer unique opportunities to illuminate the chemical intercommunications between Bacillus and plants, to elucidate biochemical and molecular details on modes of action of Bacillus-based formulations, to generate more actionable insights on cellular and molecular events that explain the Bacillus-induced growth promotion and stress resilience in plants

Selection and evaluation of housekeeping genes as endogenous controls for quantification of mRNA transcripts in Theileria parva using quantitative real-time polymerase chain reaction (qPCR).

The reliability of any quantitative real-time polymerase chain reaction (qPCR) experiment can be seriously compromised by variations between samples as well as between PCR runs. This usually result from errors in sample quantification, especially with samples that are obtained from different individuals and tissues and have been collected at various time intervals. Errors also arise from differences in qPCR efficiency between assays performed simultaneously to target multiple genes on the same plate. Consequently, the derived quantitative data for the target genes become distorted. To avoid this grievous error, an endogenous control, with relatively constant transcription levels in the target individual or tissue, is included in the qPCR assay to normalize target gene expression levels in the analysis. Several housekeeping genes (HKGs) have been used as endogenous controls in quantification studies of mRNA transcripts; however, there is no record in the literature of the evaluation of these genes for the tick-borne protozoan parasite, Theileria parva. Importantly, the expression of these genes should be invariable between different T. parva stocks, ideally under different experimental conditions, to gain extensive application in gene expression studies of this parasite. Thus, the expression of several widely used HKGs was evaluated in this study, including the genes encoding β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 28S rRNA, cytochrome b and fructose-2.6-biphosphate aldolase (F6P) proteins. The qPCR analysis revealed that the expression of genes encoding cytochrome b, F6P and GAPDH varied considerably between the two T. parva stocks investigated, the cattle-derived T. parva Muguga and the buffalo-derived T. parva 7014. 28S rRNA and β-actin gene expression was the most stable; thus, these genes were considered suitable candidates to be used as endogenous control genes for mRNA quantification studies in T. parva

Inter-assay variations analysis as determined by Student’s <i>t</i>-test in expression of the five candidate reference genes for <i>T</i>. <i>parva</i> Muguga and 7014.

<p>Inter-assay variations analysis as determined by Student’s <i>t</i>-test in expression of the five candidate reference genes for <i>T</i>. <i>parva</i> Muguga and 7014.</p

Gene expression stability of five candidate <i>T</i>. <i>parva</i> reference genes as assessed by RefFinder.

<p>Gene expression stability of five candidate <i>T</i>. <i>parva</i> reference genes as assessed by RefFinder.</p

The comparison of expression profiles of the three differentially expressed genes based on the log2 fold change values obtained from qPCR analysis when the data was normalized using 28S rRNA or β-actin as endogenous gene controls.

<p>The comparison of expression profiles of the three differentially expressed genes based on the log2 fold change values obtained from qPCR analysis when the data was normalized using 28S rRNA or β-actin as endogenous gene controls.</p

Specificity of real-time PCR amplification: Melting curves generated after amplification of five candidate <i>T</i>. <i>parva</i> reference genes showing a single melting peak for each product.

<p>Each experiment included two biological replicates of cDNA prepared from RNA isolated from cell cultures infected with <i>T</i>. <i>parva</i> Muguga and <i>T</i>. <i>parva</i> 7014 and a no template control (NTC).</p

Student’s <i>t</i>-test results for analysis of intra-assay variations in the expression of HKGs between <i>T</i>. <i>parva</i> Muguga and 7014.

<p>The results from the first run are presented in panel A and results from the second run in panel B.</p

A standard curve generated by amplification of the VP2 gene from a 10-fold dilution series of BTV cDNA of known concentration (1000 to 0.1ng/μl), used to determine the concentration of <i>T</i>. <i>parva</i> cDNA by comparison with qPCR amplification of <i>T</i>. <i>parva</i> 28S rRNA from parasite isolates <i>T</i>. <i>parva</i> Muguga (designated with a red triangle) and <i>T</i>. <i>parva</i> 7014 (designated with a green triangle).

<p>A standard curve generated by amplification of the VP2 gene from a 10-fold dilution series of BTV cDNA of known concentration (1000 to 0.1ng/μl), used to determine the concentration of <i>T</i>. <i>parva</i> cDNA by comparison with qPCR amplification of <i>T</i>. <i>parva</i> 28S rRNA from parasite isolates <i>T</i>. <i>parva</i> Muguga (designated with a red triangle) and <i>T</i>. <i>parva</i> 7014 (designated with a green triangle).</p