Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process

Analyses of different plant stressors are often based on gene expression studies. Quantitative real-time RT-PCR (qRT-PCR) is the most sensitive method for the detection of low abundance transcripts. However, a critical point to note is the selection of housekeeping genes as an internal control. Many so-called ‘housekeeping genes’ are often affected by different stress factors and may not be suitable for use as an internal reference. We tested six housekeeping genes of European beech by qRT-PCR using the Sybr Green PCR kit. Specific primers were designed for 18S rRNA, actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH1, GAPDH2), α-tubulin, and ubiquitin-like protein. Beech saplings were treated with increased concentrations of either ozone or CO2. In parallel, the expression of these genes was analyzed upon pathogen infection with Phytophthora citricola. To test the applicability of these genes as internal controls under realistic outdoor conditions, sun and shade leaves of 60-year-old trees were used for comparison. The regulation of all genes was tested using a linear mixed-effect model of the R-system. Results from independent experiments showed that the only gene not affected by any treatment was actin. The expression of the other housekeeping genes varied more or less with the degree of stress applied. These results highlight the importance of undergoing an individual selection of internal control genes for different experimental conditions.

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Selection of Suitable Internal Control Genes for Accurate Normalization of Real-Time Quantitative PCR Data of Buffalo (Bubalus bubalis) Blastocysts Produced by SCNT and IVF

Cellular Reprogramming ◽

10.1089/cell.2017.0010 ◽

2017 ◽

Vol 19 (5) ◽

pp. 302-310 ◽

Cited By ~ 5

Author(s):

Tanushri Jerath Sood ◽

Swati Viviyan Lagah ◽

Ankita Sharma ◽

Suresh Kumar Singla ◽

Manishi Mukesh ◽

...

Keyword(s):

Real Time ◽

Quantitative Pcr ◽

Internal Control ◽

Bubalus Bubalis ◽

Real Time Quantitative Pcr ◽

Internal Control Genes ◽

Accurate Normalization ◽

Selection Of

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Investigation of Different Commercially Available Real Time-Polymerase Chain Reaction Kits for SARS-CoV-2 Diagnosis

JOURNAL FOR CLINICAL AND DIAGNOSTIC RESEARCH ◽

10.7860/jcdr/2021/48726.14977 ◽

2021 ◽

Author(s):

Rajeev Kumar Jain ◽

Nagaraj Perumal ◽

Rakesh Shrivastava ◽

Kamlesh Kumar Ahirwar ◽

Jaya Lalwani ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Internal Control ◽

Housekeeping Genes ◽

Housekeeping Gene ◽

Specific Gene ◽

Rt Pcr ◽

Chain Reaction ◽

Gene Target ◽

Polymerase Chain

Introduction: The whole world is facing an ongoing global health emergency of COVID-19 disease caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is a gold standard in the detection of SARS-CoV-2 infection. Presently, many single tube multiple gene target RT-PCR kits have been developed and are commercially available for Coronavirus Disease 2019 (COVID-19) diagnosis. Aim: To evaluate the performance of seven COVID-19 RT-PCR kits (DiagSure, Meril, VIRALDTECT II, TruPCR, Q-line, Allplex and TaqPath) which are commercially available for COVID-19 RT-PCR diagnosis. Materials and Methods: This observational study was conductedat the State Virology Laboratory (SVL), Gandhi Medical College, Bhopal, Madhya Pradesh, India. Seven commercially available kits have been evaluated on the basis of: (i) number of SARS-CoV-2 specific gene target; (ii) human housekeeping genes as internal control; (iii) RT-PCR run time; and (iv) kit performances to correctly detect SARS-CoV-2 positive and negative RNA samples. A total of 50 RNA samples (left over RNA) were included, master mix preparation, template addition and RT-PCR test has been performed according to kits literature. At the end of PCR run, mean and standard deviation of obtained cut-off of all kits were calculated using Microsoft Excel. Results: All seven RT-PCR kits performed satisfactory regarding the reproducibility and they could correctly identify 30 positive and 20 negative RNA samples. RNA samples (group C) having low viral loads with a high Cycle threshold (Ct) value (>30) were also detected by all these seven kits. Obtained Ct values of each group was in parallel range in comparison with the initial testing Ct values. Kits were found to be superior which contains primers and probes for three SARS-CoV-2 specific gene targets, have human housekeeping gene as internal control and taking less time to complete RT-PCR. Conclusion: All seven COVID-19 RT-PCR kits included in this study demonstrated satisfactory performance and can be used for the routine molecular diagnosis of COVID-19 disease.

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Developing Online Materials for Tour Guides

Journal of ELT Research ◽

10.22236/jer_vol3issue1pp43-57 ◽

2018 ◽

Vol 3 (1) ◽

pp. 43

Author(s):

Sinta Dewi Ratnawati

Keyword(s):

Online Learning ◽

Real Time ◽

Development Process ◽

Language Teaching ◽

Teaching Approach ◽

Online Classes ◽

Material Development ◽

Tour Guides ◽

Goals And Objectives ◽

Selection Of

This English for Specific/Occupational Purpose (ESP/EOP) program was meant to answer the needs of the Indonesian migrant labourers to improve their English for future employment as tour guides. The objectives of this research were to identify the contents that should be in the materials, to investigate the students’ response about the later developed materials, and to study the lacks in the program. The material development process underwent the 7 stages of material development proposed by Nation and Macalister (2010), consisted of analysing the environment and the needs, following principles, setting goals and objectives, choosing contents and sequence, finding the format and presentation, monitoring and assessing, and evaluating the course. However, since it is online learning, the stages had to be combined with the stages specifically for online materials proposed by Hartoyo (2012). Thus, the content and sequence choosing stage consisted of the selection of the types of program, materials, software and tasks. The materials consisted of 2 parts which were the tutorials and the lessons. Using the Task-Based Language Teaching approach (Nunan, 2004), the materials were developed and implemented combined with real time online classes. The students’ responses after the implementation were generally positive, although some improvements were still needed. Keywords: English for specific purpose (ESP), English for occupational purpose (EOP), material development, online learning, English for tour guides, Task-Based Language Teaching (TBLT)

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