Dataset Information

Validation of Reference Genes for Quantitative Real-Time PCR in Bovine PBMCs Transformed and Non-transformed by Theileria annulata.

ABSTRACT: Theileria annulata is a tick-borne intracellular protozoan parasite that causes tropical theileriosis, a fatal bovine lymphoproliferative disease. The parasite predominantly invades bovine B lymphocytes and macrophages and induces host cell transformation by a mechanism that is not fully comprehended. Analysis of signaling pathways by quantitative real-time PCR (qPCR) could be a highly efficient means to understand this transformation mechanism. However, accurate analysis of qPCR data relies on selection of appropriate reference genes for normalization, yet few papers on T. annulata contain evidence of reference gene validation. We therefore used the geNorm and NormFinder programs to evaluate the stability of 5 candidate reference genes; 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ACTB (?-actin), PRKG1 (protein kinase cGMP-dependent, type I) and TATA box binding protein (TBP). The results showed that 18S rRNA was the reference gene most stably expressed in bovine PBMCs transformed and non-transformed with T. annulata, followed by GAPDH and TBP. While 18S rRNA and GAPDH were the best combination, these 2 genes were chosen as references to study signaling pathways involved in the transformation mechanism of T. annulata.

SUBMITTER: Zhao H

PROVIDER: S-EPMC4792322 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Validation of Reference Genes for Quantitative Real-Time PCR in Bovine PBMCs Transformed and Non-transformed by Theileria annulata.

Zhao Hongxi H Liu Junlong J Li Youquan Y Yang Congshan C Zhao Shuaiyang S Liu Juan J Liu Aihong A Liu Guangyuan G Yin Hong H Guan Guiquan G Luo Jianxun J

The Korean journal of parasitology 20160226 1

Theileria annulata is a tick-borne intracellular protozoan parasite that causes tropical theileriosis, a fatal bovine lymphoproliferative disease. The parasite predominantly invades bovine B lymphocytes and macrophages and induces host cell transformation by a mechanism that is not fully comprehended. Analysis of signaling pathways by quantitative real-time PCR (qPCR) could be a highly efficient means to understand this transformation mechanism. However, accurate analysis of qPCR data relies on ...[more]

PMID: 26951977

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Project description:BACKGROUND:The use of stably expressed genes as normalizers has crucial role in accurate and reliable expression analysis estimated by quantitative real-time polymerase chain reaction (qPCR). Recent studies have shown that, the expression levels of common housekeeping genes are varying in different tissues and experimental conditions. The genomic DNA contamination in RNA samples is another important factor that also influence the interpretation of the data obtained from qPCR. It is estimated that the gDNA contamination in gene expression analysis lead to an overestimation of the RNA transcript level. The aim of this study was to validate the most stably expressed reference genes in two different tissues of Aeluropus littoralis-halophyte grass at salt stress and recovery condition. Also, a qPCR-based approach for monitoring contamination with gDNA was conducted. RESULTS:Ten candidate reference genes participating in different biological processes were analyzed in four groups of samples including root and leaf tissues, salt stress and recovery condition. To determine the most stably expressed reference genes, three statistical methods (geNorm, NormFinder and BestKeeper) were applied. According to results obtained, ten candidate reference genes were ranked based on the stability of their expression. Here, our results show that a set of four housekeeping genes (HKGs) e.g. RPS3, EF1A, GTF and RPS12 could be used as general reference genes for the all selected conditions and tissues. Also, four set of reference genes were proposed for each tissue and condition including: RPS3, EF1A and UBQ for salt stress and root samples; RPS3, EF1A, UBQ as well as GAPDH for recovery condition; U2SURP and GTF for leaf samples. Additionally, for assessing DNA contamination in RNA samples, a set of unique primers were designed based on the conserved region of ribosomal DNA (rDNA). The universality, specificity and sensitivity of these primer pairs were also evaluated in Poaceae. CONCLUSIONS:Overall, the sets of reference genes proposed in this study are ideal normalizers for qPCR analysis in A. littoralis transcriptome. The novel reference gene e.g. RPS3 that applied this study had higher expression stability than commonly used housekeeping genes. The application of rDNA-based primers in qPCR analysis was addressed.

Project description:BackgroundWith the availability of genome sequences, gene expression analysis of jute has drawn considerable attention for understanding the regulatory mechanisms of fiber development and improving fiber quality. Gene expression profiles of a target gene can provide valuable clues towards the understanding of its biological function. Reverse transcription quantitative real-time PCR (qRT-PCR) is the best method for targeted gene expression analysis due to its sensitivity and reproducibility. However, calculating relative expression requires reference genes, which must be stable across various biological conditions. For this purposes, 11 prospective genes namely, 28S RNA, ACT7, CYP, EF1A, EF2, ETIF3E, GAPDH, PP2Ac, PTB, UBC2 and UBI1 were evaluated for their potential use as reference genes in jute.ResultsThe expression stabilities of eleven prospective genes were analyzed in various jute plant tissues, such as the root, stick, bark, leaf, flower, seed and fiber, as well as under abiotic (waterlogged, drought and salinity) and biotic stress (infestation with Macrophomina phaseolina) conditions with different time points. All 11 genes were variably expressed in different tissues and stress conditions. To find suitable reference genes in different sample sets, a comprehensive approach based on four statistical algorithms such as GeNorm, BestKeeper, NormFinder the ?Ct was used. The PP2Ac and EF2 genes were the most stably expressed across the different tissues. ACT7 and UBC2 were suitable reference genes under drought stress, and CYP and PP2Ac were the most appropriate after inoculation with Macrophomina phaseolina. Under salinity stress, PP2Ac and UBC2 were the best genes, and ACT7 and PP2Ac were the most suitable under waterlogged conditions.ConclusionExpression stability of reference genes from jute varied in different tissues and selected experimental conditions. Our results provide a valuable resource for the accurate normalization of gene expression experiments in fiber research for important bast fiber crops.

Dataset Information

Validation of Reference Genes for Quantitative Real-Time PCR in Bovine PBMCs Transformed and Non-transformed by Theileria annulata.

Publications

Validation of Reference Genes for Quantitative Real-Time PCR in Bovine PBMCs Transformed and Non-transformed by Theileria annulata.

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