Selection of Internal Reference Genes for Normalization of Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) Analysis in the Canine Brain and Other Organs

2012 ◽  
Vol 54 (1) ◽  
pp. 47-57 ◽  
Author(s):  
Sang-Je Park ◽  
Jae-Won Huh ◽  
Young-Hyun Kim ◽  
Sang-Rae Lee ◽  
Sang-Hyun Kim ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Reference Genes ◽  
Pcr Analysis ◽  
Chain Reaction ◽  
Internal Reference ◽  
Qrt Pcr ◽  
Canine Brain ◽  
Polymerase Chain ◽  
Selection Of

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 Reference Gene Selection for Real-time Quantitative Reverse-transcription Polymerase Chain Reaction in Flower Buds of Marigold

2021 ◽  
pp. 1-13
Author(s):  
Nan Tang ◽  
Wuhua Zhang ◽  
Liwen Chen ◽  
Yan Wang ◽  
Daocheng Tang
Keyword(s):  
Polymerase Chain Reaction ◽  
Male Sterility ◽  
Reverse Transcription ◽  
Reference Genes ◽  
Male Sterile ◽  
Chain Reaction ◽  
Flower Buds ◽  
Qrt Pcr ◽  
Polymerase Chain ◽  
Suitable Reference

Marigold (Tagetes erecta) is an important commercial plant because of its ornamental, industrial, and medicinal values. Male-sterile two-type lines are important for heterosis utilization and breeding of marigold. Mining of fertility-related genes may help to elucidate the mechanisms underlying male sterility. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a popular and useful tool for analyzing the expression level of a specific gene. Notably, identifying a suitable reference gene is important for data normalization because it affects the accuracy of quantitative analysis. However, at present, no reference genes are available for marigold. During the current study, 10 candidate reference genes were selected and their expression levels in different samples were analyzed by qRT-PCR. The expression level of each gene was analyzed across different developmental stages of male-sterile and male-fertile flower buds by four software programs (geNorm, NormFinder, BestKeeper, and RefFinder). The results showed that different reference genes are required for male-sterile and male-fertile samples, even if they belong to the same line. For male-sterile samples, the ribosomal protein S5/18S ribosomal RNA (RPS5/18S) gene pair was the best reference for qRT-PCR normalization, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) could be used as an alternative. For male-fertile samples, elongation factor 1-alpha (EF1α) and RPS5 were the most suitable reference genes, and Ubiquitin-conjugating enzyme (UBC) could be used as an alternative. Beta-actin (ACTB), tubulin beta (TUB), and phenylalanine ammonia-lyase (PAL) should not be used as reference genes because they were the most unstable genes in flower buds of marigold. The results of the current study may facilitate the selection of reference genes for analyzing the expression patterns of genes involved in flower development related to male sterility in marigold.

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