scholarly journals A probe-based qRT-PCR method to profile immunological gene expression in blood of captive beluga whales (Delphinapterus leucas)

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3840 ◽  
Author(s):  
Ming-An Tsai ◽  
I-Hua Chen ◽  
Jiann-Hsiung Wang ◽  
Shih-Jen Chou ◽  
Tsung-Hsien Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune System ◽  
Reference Genes ◽  
Animal Health ◽  
Rna Extraction ◽  
Disease Onset ◽  
Delphinapterus Leucas ◽  
Amplification Efficiency ◽  
Beluga Whales ◽  
Qrt Pcr

Cytokines are fundamental for a functioning immune system, and thus potentially serve as important indicators of animal health. Quantitation of mRNA using quantitative reverse transcription polymerase chain reaction (qRT-PCR) is an established immunological technique. It is particularly suitable for detecting the expression of proteins against which monoclonal antibodies are not available. In this study, we developed a probe-based quantitative gene expression assay for immunological assessment of captive beluga whales (Delphinapterus leucas) that is one of the most common cetacean species on display in aquariums worldwide. Six immunologically relevant genes (IL-2Rα, -4, -10, -12, TNFα, and IFNγ) were selected for analysis, and two validated housekeeping genes (PGK1 and RPL4) with stable expression were used as reference genes. Sixteen blood samples were obtained from four animals with different health conditions and stored in RNAlater™ solution. These samples were used for RNA extraction followed by qRT-PCR analysis. Analysis of gene transcripts was performed by relative quantitation using the comparative Cq method with the integration of amplification efficiency and two reference genes. The expression levels of each gene in the samples from clinically healthy animals were normally distributed. Transcript outliers for IL-2Rα, IL-4, IL-12, TNFα, and IFNγ were noticed in four samples collected from two clinically unhealthy animals. This assay has the potential to identify immune system deviation from normal state, which is caused by health problems. Furthermore, knowing the immune status of captive cetaceans could help both trainers and veterinarians in implementing preventive approaches prior to disease onset.

scholarly journals A novel universal probe library quantitative reverse transcription polymerase chain reaction method to profile immunological gene expression in blood of captive beluga whales (Delphinapterusleucas)

2017 ◽  
Author(s):  
Ming-An Tsai ◽  
I-Hua Chen ◽  
Jiann-Hsiung Wang ◽  
Shih-Jen Chou ◽  
Tsung-Hsien Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Polymerase Chain Reaction ◽  
Immune System ◽  
Reverse Transcription ◽  
Reference Genes ◽  
Amplification Efficiency ◽  
Chain Reaction ◽  
Beluga Whales ◽  
Qrt Pcr ◽  
Polymerase Chain

Cytokines are fundamental for a functioning immune system, and thus, potentially serve as important indicators of animal health. Quantitation of mRNA using quantitative reverse transcription polymerase chain reaction (qRT-PCR) is an established immunological technique. It is particularly suitable for detecting the expression of proteins against which monoclonal antibodies are not available. In this study, we developed a novel probe-based quantitative gene expression assay for immunological assessment of captive beluga whales ( Delphinapterusleucas ) that is one of the most common cetacean species on display in aquariums worldwide. Six immunologically relevant genes (IL-2Rα, -4, -10, -12, TNFα, and IFNγ) were selected for analysis, and two validated housekeeping genes (PGK1 and RPL4) with stable expression were used as reference genes. Sixteen blood samples were obtained from four animals with different health conditions and stored in RNAlater solution. These samples were used for RNA extraction followed by qRT-PCR analysis. Analysis of gene transcripts was performed by relative quantitation using the comparative Cq method with the integration of amplification efficiency and two reference genes. The expression levels of each gene in the samples from clinically healthy animals were normally distributed. Transcript outliers for IL-2Rα, IL-4, IL-12, TNFα, and IFNγ were noticed in four samples collected from two clinically unhealthy animals. This assay has the potential to identify immune system deviation from normal state, which is caused by health problems. Furthermore, knowing the immune status of captive cetaceans could help both trainers and veterinarians in implementing preventive approaches prior to disease onset.

scholarly journals A novel universal probe library quantitative reverse transcription polymerase chain reaction method to profile immunological gene expression in blood of captive beluga whales (Delphinapterusleucas)

2017 ◽  
Author(s):  
Ming-An Tsai ◽  
I-Hua Chen ◽  
Jiann-Hsiung Wang ◽  
Shih-Jen Chou ◽  
Tsung-Hsien Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Polymerase Chain Reaction ◽  
Immune System ◽  
Reverse Transcription ◽  
Reference Genes ◽  
Amplification Efficiency ◽  
Chain Reaction ◽  
Beluga Whales ◽  
Qrt Pcr ◽  
Polymerase Chain

Cytokines are fundamental for a functioning immune system, and thus, potentially serve as important indicators of animal health. Quantitation of mRNA using quantitative reverse transcription polymerase chain reaction (qRT-PCR) is an established immunological technique. It is particularly suitable for detecting the expression of proteins against which monoclonal antibodies are not available. In this study, we developed a novel probe-based quantitative gene expression assay for immunological assessment of captive beluga whales ( Delphinapterusleucas ) that is one of the most common cetacean species on display in aquariums worldwide. Six immunologically relevant genes (IL-2Rα, -4, -10, -12, TNFα, and IFNγ) were selected for analysis, and two validated housekeeping genes (PGK1 and RPL4) with stable expression were used as reference genes. Sixteen blood samples were obtained from four animals with different health conditions and stored in RNAlater solution. These samples were used for RNA extraction followed by qRT-PCR analysis. Analysis of gene transcripts was performed by relative quantitation using the comparative Cq method with the integration of amplification efficiency and two reference genes. The expression levels of each gene in the samples from clinically healthy animals were normally distributed. Transcript outliers for IL-2Rα, IL-4, IL-12, TNFα, and IFNγ were noticed in four samples collected from two clinically unhealthy animals. This assay has the potential to identify immune system deviation from normal state, which is caused by health problems. Furthermore, knowing the immune status of captive cetaceans could help both trainers and veterinarians in implementing preventive approaches prior to disease onset.

scholarly journals Selection of reference genes for RT-qPCR studies in blood of beluga whales (Delphinapterus leucas)

2016 ◽  
Author(s):  
I-Hua Chen ◽  
Jiann-Hsiung Wang ◽  
Shih-Jen Chou ◽  
Yeong-Huey Wu ◽  
Tsung-Hsien Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Gene Selection ◽  
Housekeeping Genes ◽  
Delphinapterus Leucas ◽  
Correct Result ◽  
Relative Quantification ◽  
Blood Leukocytes ◽  
Blood Samples ◽  
Beluga Whales

Reverse transcription-quantitative PCR (RT-qPCR) is used for research in gene expression, and it is vital to choose appropriate housekeeping genes (HKGs) as reference genes to obtain correct result. The purpose of this study is to determine stably expressed HKGs in blood of beluga whales (Delphinapterus leucas) that can be the appropriate reference genes in relative quantification in gene expression research. Sixty blood samples were taken from 4 beluga whales. Thirteen candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ) were tested using RT-qPCR. The stability values of the HKGs were determined by four different algorithms. Comprehensive analysis of the results revealed that RPL4, PGK1 and ACTB are strongly recommended for use in future RT-qPCR studies in beluga blood samples. This research provides recommendation of reference gene selection, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans. The gene expression assessment of the immune components in blood have potential to serve as important approach to evaluating cetacean health influenced by environmental insults.

scholarly journals Selection of reference genes for RT-qPCR studies in blood of beluga whales (Delphinapterus leucas)

2016 ◽  
Author(s):  
I-Hua Chen ◽  
Jiann-Hsiung Wang ◽  
Shih-Jen Chou ◽  
Yeong-Huey Wu ◽  
Tsung-Hsien Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Gene Selection ◽  
Housekeeping Genes ◽  
Delphinapterus Leucas ◽  
Correct Result ◽  
Relative Quantification ◽  
Blood Leukocytes ◽  
Blood Samples ◽  
Beluga Whales

Reverse transcription-quantitative PCR (RT-qPCR) is used for research in gene expression, and it is vital to choose appropriate housekeeping genes (HKGs) as reference genes to obtain correct result. The purpose of this study is to determine stably expressed HKGs in blood of beluga whales (Delphinapterus leucas) that can be the appropriate reference genes in relative quantification in gene expression research. Sixty blood samples were taken from 4 beluga whales. Thirteen candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ) were tested using RT-qPCR. The stability values of the HKGs were determined by four different algorithms. Comprehensive analysis of the results revealed that RPL4, PGK1 and ACTB are strongly recommended for use in future RT-qPCR studies in beluga blood samples. This research provides recommendation of reference gene selection, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans. The gene expression assessment of the immune components in blood have potential to serve as important approach to evaluating cetacean health influenced by environmental insults.

scholarly journals Identification of Reference Genes for Quantitative Gene Expression Studies in Pinus massoniana and Its Introgression Hybrid

Forests ◽  
2019 ◽  
Vol 10 (9) ◽  
pp. 787
Author(s):  
Jiaxing Mo ◽  
Jin Xu ◽  
Wenjing Jin ◽  
Liwei Yang ◽  
Tongming Yin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
Gene Selection ◽  
Regulation Of Gene Expression ◽  
Threshold Value ◽  
Pinus Massoniana ◽  
Tissue Type ◽  
Amplification Efficiency ◽  
Qrt Pcr

qRT-PCR is a powerful molecular research tool to study the regulation of gene expression. However, to accurately calculate gene expression levels, an experiment should include proper reference genes that show no changes in their expression level. Pinus massoniana, P. hwangshanensis, and their introgression hybrid in Mountain Lushan, China, are an ideal model for studying introgression and speciation. Although some research on reference gene selection for P. massoniana has been reported before, no studies on this subject have been performed where P. massoniana and its introgression hybrid were evaluated simultaneously. Here, we investigated ten genes (upLOC, SDH, ACT, EF, TOC75, DMWD, FBOX, PGK1, UBQ, and CL2417C7) identified from transcriptome data of these two taxa for reference gene potential. These ten genes were then screened across multiple tissues such as cone, young and mature stems, and young needles according to qRT-PCR thermal cycling and dissociation. Correlation coefficient, amplification efficiency, and cycle threshold value (Ct) range were applied to evaluate the reliability of each gene. The stability of candidate reference gene expression was calculated using three algorithms: geNorm, NormFinder, and BestKeeper. Base on the reliability and stability, we then offered a list of genes of recommended and not recommended for seven different tissue type and species. Our results demonstrated that different sample lines require different genes as reference to evaluate.

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