scholarly journals Selection of Suitable Reference Genes for Quantitative Real-time Polymerase Chain Reaction in Prunus mume during Flowering Stages and under Different Abiotic Stress Conditions

2014 ◽  
Vol 139 (2) ◽  
pp. 113-122 ◽  
Author(s):  
Tao Wang ◽  
Ruijie Hao ◽  
Huitang Pan ◽  
Tangren Cheng ◽  
Qixiang Zhang
Keyword(s):  
Polymerase Chain Reaction ◽  
Abiotic Stress ◽  
Real Time ◽  
Reference Genes ◽  
Late Embryogenesis Abundant Protein ◽  
Prunus Mume ◽  
Chain Reaction ◽  
Qrt Pcr ◽  
Polymerase Chain ◽  
Suitable Reference

Mei (Prunus mume) is widely cultivated in eastern Asia owing to its favored ornamental characteristics and its tolerance for low temperatures. Reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used method for gene expression analysis, requiring carefully selected reference genes to ensure data reliability. The aim of this study was to identify and evaluate reference genes for qRT-PCR in mei. Ten candidate reference genes were chosen, and their expression levels were assessed by qRT-PCR in four sample sets: 1) flowering mei; 2) mei undergoing abiotic stress; 3) different genotypes of Prunus species; and 4) all mei samples. The stability and suitability of the candidate reference genes were validated using commercially available software. We found that protein phosphatase 2A-1 (PP2A-1) and PP2A-2 were suitable reference genes for flowering with ubiquitin-conjugating enzyme E2 (UBC) also being suitable for different genotypes of Prunus species. UBC and actin (ACT) were most stably expressed under abiotic stress. Finally, the expression of an AGAMOUS homolog of Arabidopsis thaliana (PmAG) and a putative homolog of Group 2 late embryogenesis abundant protein gene in A. thaliana (PmLEA) were assessed to allow comparisons between selected candidate reference genes, highlighting the importance of careful reference gene selection.

scholarly journals Identification of suitable reference genes in blood samples of carcinoma lung patients using quantitative real-time polymerase chain reaction

2020 ◽  
Vol 19 (1) ◽  
pp. 11
Author(s):  
Sweety Gupta ◽  
ShashiRanjan Mani Yadav ◽  
Bela Goyal ◽  
Raman Kumar ◽  
Amit Gupta ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Reference Genes ◽  
Chain Reaction ◽  
Blood Samples ◽  
Polymerase Chain ◽  
Suitable Reference

scholarly journals Identification of Suitable Reference Genes for Real Time Quantitative Polymerase Chain Reaction Assays on Pectoralis major Muscle in Chicken (Gallus gallus )

PLoS ONE ◽  
2015 ◽  
Vol 10 (5) ◽  
pp. e0127935 ◽  
Author(s):  
Carlos S. Nascimento ◽  
Leandro T. Barbosa ◽  
Claudson Brito ◽  
Roberta P. M. Fernandes ◽  
Renata S. Mann ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Reference Genes ◽  
Quantitative Polymerase Chain Reaction ◽  
Pectoralis Major Muscle ◽  
Gallus Gallus ◽  
Pectoralis Major ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Suitable Reference

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.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

Validation of Reference Genes for Real-Time Polymerase Chain Reaction Studies in Atlantic Salmon

2006 ◽  
Vol 8 (4) ◽  
pp. 398-408 ◽  
Author(s):  
Sven Martin Jorgensen ◽  
Ellen Johanne Kleveland ◽  
Unni Grimholt ◽  
Tor Gjoen
Keyword(s):  
Polymerase Chain Reaction ◽  
Atlantic Salmon ◽  
Real Time ◽  
Reference Genes ◽  
Chain Reaction ◽  
Polymerase Chain
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