26S rRNA-based internal control gene primer pair for reverse transcription-polymerase chain reaction-based quantitative expression studies in diverse plant species

2004 ◽  
Vol 335 (2) ◽  
pp. 330-333 ◽  
Author(s):  
Kashmir Singh ◽  
Jyoti Raizada ◽  
Pradeep Bhardwaj ◽  
Sanjay Ghawana ◽  
Arti Rani ◽  
...  
Keyword(s):  
Internal Control ◽  
Control Gene ◽  
Internal Control Gene ◽  
Chain Reaction ◽  
Quantitative Expression ◽  
Expression Studies ◽  
Polymerase Chain ◽  
26S Rrna ◽  
Diverse Plant Species ◽  
Diverse Plant

scholarly journals Transcriptional expression study in the central nervous system of rats: what gene should be used as internal control?

2014 ◽  
Vol 12 (3) ◽  
pp. 336-341 ◽  
Author(s):  
Ana Carolina de Moura ◽  
Virgínia Meneghini Lazzari ◽  
Grasiela Agnes ◽  
Silvana Almeida ◽  
Márcia Giovenardi ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Internal Control ◽  
Housekeeping Genes ◽  
Internal Control Gene ◽  
Chain Reaction ◽  
Beta Actin ◽  
The Central Nervous System ◽  
Polymerase Chain ◽  
The Brain

Objective A growing number of published articles report the expression of specific genes with different behavior patterns in rats. The levels of messenger ribonucleic acid transcripts are usually analyzed by reverse transcription followed by polymerase chain reaction and quantified after normalization with an internal control or reference gene (housekeeping gene). Nevertheless, housekeeping genes exhibit different expression in the central nervous system, depending on the physiological conditions and the area of the brain to be studied. The choice of a good internal control gene is essential for obtaining reliable results. This study evaluated the expression of three housekeeping genes (beta-actin, cyclophilin A, and ubiquitin C) in different areas of the central nervous system in rats (olfactory bulb, hippocampus, striatum, and prefrontal cortex). Methods Wistar rats (virgin females, n=6) during the diestrum period were used. Total ribonucleic acid was extracted from each region of the brain; the complementary deoxyribonucleic acid was synthesized by reverse transcription and amplified by real-time quantitative polymerase chain reaction using SYBR™ Green and primers specific for each one of the reference genes. The stability of the expression was determined using NormFinder. Results Beta-actin was the most stable gene in the hippocampus and striatum, while cyclophilin A and ubiquitin C showed greater stability in the prefrontal cortex and the olfactory bulb, respectively. Conclusion Based on our study, further studies of gene expression using rats as animal models should take into consideration these results when choosing a reliable internal control gene.

scholarly journals Genotyping of Listeria by Polymerase Chain Reaction (PCR) and its Epidemiological Significance

2021 ◽  
Vol 6 (2) ◽  
pp. 115-124
Author(s):  
V. Zyuzin ◽  
◽  
O. Tuzova ◽  
U. Frenkel ◽  
Muntian L. ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Internal Control ◽  
Pcr Analysis ◽  
Chain Reaction ◽  
Virulence Markers ◽  
Chip Technology ◽  
Polymerase Chain ◽  
Primer Sets ◽  
Epidemiological Significance

The purpose of the study. The article covers the issues of genotyping of listeria by polymerase chain reaction (PCR) and its epidemiological significance. It is known that molecular genetic methods allow to detect specific microbial pathogens, virulence markers, antimicrobial resistance genes faster and with greater sensitivity than traditional culture methods. Therefore, the development of detection methods and genotyping by polymerase chain reaction (PCR) is relevant. Materials and methods. For the detection and genotyping of Listeria, the technology of DNA chips is becoming increasingly important, which can significantly expand the possibilities of molecular detection. Chip technology can be used to simultaneously identify a whole range of pathogenic microorganisms, to determine genetic virulence markers, the relationship to antibiotics, subtyping, as well as to determine the quality of microorganisms in samples. A simplified version of DNA chip technology is multiplex (numerical) PCR, which is used to detect and genotype listeria. Studies have shown that to detect Listeria spp. using a polymerase chain reaction, it is advisable to use the gene iap (invasive associated protein), known for 6 species of listeria, which encodes a protein P 60 that is common to all species of listeria, including L.murrayi. Computer analysis revealed areas with 100% homology, from which primers were selected for PCR detection of all types of listeria. Areas of genomes characterized by 100% homology were selected for further analysis and labeling of primer sets. The sequences of the constructed primers List 1 and List 2 allowed to identify 6 species of Listeria (L. monocytogenes, L. innocua, L. ivanovii, L. grayi, L. seeligeri, L. welshimeri). Increasing the length of the primer leads to the increasing of specificity of PCR analysis. The greater the length of the primer, the smaller the specific gravity of one error of the unpaired nucleotide. The degree of primer homology is a key parameter that indicates the "quality" of a set of primers. Results and discussion. It is established that a significant disadvantage of the vast majority diagnosed using PCR test systems is the lack of internal control of amplification. The negative result of PCR analysis may be due to the absence in the clinical material of a fragment of the Listeria genome, and the fact that the PCR product was not synthesized for other reasons. They may be as the following ones: operator errors, erroneously determined reaction mixture concentrations and PCR temperature parameters. False-negative results can also be caused by factors that inhibit thermostable DNA polymerase. In its turn, such inhibition of the enzyme responsible for amplification is caused by a very large amount of DNA - template, pre-treatment of clinical samples. It has been shown that 80% of clinical specimens contain a substance that inhibits DNA polymerase. Therefore, it is necessary to use internal control, the positive result of the reaction of which indicates the successful amplification, that is the absence of false positive results. Conclusion. There are several reasons why the accuracy of PCR analysis does not reach 100%. Accuracy depends on the technology (variety) of PCR - the method used (ordinary or fluorescent), detection of amplicons, PCR homogeneous or nested, nested in one test tube or in two test tubes, as well as the level of quality of the survey (primarily on the technical parameters of the amplifier). The test systems used can be used for PCR detection and are recommended as standard primer sets for the detection and cross-species testing of listeria, which is important for the timely implementation of appropriate anti-epidemic measures in listeriosis

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

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.

scholarly journals β-glucuronidase use as a single internal control gene may confound analysis in FMR1 mRNA toxicity studies

PLoS ONE ◽  
2018 ◽  
Vol 13 (2) ◽  
pp. e0192151 ◽  
Author(s):  
Claudine M. Kraan ◽  
Kim M. Cornish ◽  
Quang M. Bui ◽  
Xin Li ◽  
Howard R. Slater ◽  
...  
Keyword(s):  
Internal Control ◽  
Control Gene ◽  
Internal Control Gene ◽  
Toxicity Studies ◽  
Fmr1 Mrna

Utility of an internal control for the polymerase chain reaction

Apmis ◽  
1992 ◽  
Vol 100 (7-12) ◽  
pp. 635-639 ◽  
Author(s):  
JEAN-PAUL URSI ◽  
DOMINIQUE URSI ◽  
MARGARETA IEVEN ◽  
STEFAAN R. PATTYN
Keyword(s):  
Polymerase Chain Reaction ◽  
Internal Control ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics

2017 ◽  
Vol 112 (5) ◽  
pp. 339-347 ◽  
Author(s):  
Miriam Ribas Zambenedetti ◽  
Daniela Parada Pavoni ◽  
Andreia Cristine Dallabona ◽  
Alejandro Correa Dominguez ◽  
Celina de Oliveira Poersch ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Internal Control ◽  
Rna Viruses ◽  
Chain Reaction ◽  
Ms2 Bacteriophage ◽  
Polymerase Chain
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