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Dengue Fever: A Viral Hemorrhagic Fever of Global Concern
Published in Jagriti Narang, Manika Khanuja, Small Bite, Big Threat, 2020
Bennet Angel, Neelam Yadav, Jagriti Narang, Annette Angel, Vinod Joshi
Reverse transcriptase-polymerase chain reaction (RT-PCR): RTPCR is a well-exploited technique for the detection of RNA-based viruses. The first step is the isolation of RNA from the DENV by commercially available kits (Qiagen, Invitrogen, Ambion, etc.) or the TRIazol method (Chomczynski-Sacchi) (DePaula et al., 2002; Deubel et al., 1990; Deubel and Pierre, 1994; Figueiredo et al., 1998; Fulop et al., 1993; Lanciotti et al., 1996; Suk-Yin et al., 1994), and then it is amplified using primers. Reverse transcriptase enzyme helps in the conversion of RNA into DNA template. Three authors from the chapter have designed indigenous set of primers for all DENV serotypes (Joshi et al., 2018). DENV can be tested by using different types of PCR such as semi-nested PCR (Gomes et al., 2007), NASBA technique (Usawattankul et al., 2002), and RT-LAMP (Sahni et al., 2013).
Rabies and other lyssaviruses
Published in Avindra Nath, Joseph R. Berger, Clinical Neurovirology, 2020
Thiravat Hemachudha, Jiraporn Laothamatas, Henry Wilde
Sources for rabies virus RNA detection are saliva, extracted hair follicles or biopsied skin tissue at the nape of the neck containing hair follicles, CSF and urine [154,160–162]. Descriptions of biological samples and molecular methods have been detailed elsewhere [163]. Commonly used techniques, such as heminested reverse transcription-polymerase chain reaction (hnRT-PCR) (targeting L polymerase gene) and TaqMan real-time RT-PCR (N protein gene), have comparable degrees of sensitivity in detection; volume of tissue may be crucial [164]. hnRT-PCR assay of nuchal skin biopsy specimens containing hair follicles (approximate diameter of 4 mm; total volume of 20 mm3) is almost 100% sensitive [154]. Comparable results were obtained when at least three serial saliva samples were examined or when three types of specimens (saliva, CSF, urine or hair follicles) were assayed simultaneously [162,163]. This, however, may apply with certainty only to furious patients, since results were negative in half (3 of 6) of paralytic patients. Negative results were higher in paralytic patients than in furious; saliva samples (7/9 [77.8%] vs. 8/53 [15%]), CSF (3/5 [60%] vs. 14/25 [56%]), urine (5/5 [100%] vs. 20/36 [55.5%]), and extracted hair follicles (1/1 [100%] vs. 12/25 [48%]) [162].
Gene Expression–Based Predictors of Prognosis and Response to Chemotherapy in Breast Cancer
Published in Brian Leyland-Jones, Pharmacogenetics of Breast Cancer, 2020
There have been reports on the use of reverse transcription polymerase chain reaction (RT-PCR) to measure expression levels of few genes using RNA isolated from FFPET, but large-scale RT-PCR was not reported until recently. Cronin et al. from Genomic Health Inc. has invested on developing high quality controlled robotic assays for RT-PCR and published proof of concept study (8). In the latter study, while RNAs isolated from old formalin fixed tissue were heavily fragmented with average size under 200 base pairs and absolute signal was much less for older samples, normalization of signal using a set of reference genes could largely correct these problems.
Overview of gene expression techniques with an emphasis on vitamin D related studies
Published in Current Medical Research and Opinion, 2023
Jeffrey Justin Margret, Sushil K. Jain
The real-time, reverse transcription polymerase chain reaction (RT-PCR) is the gold standard technique used to quantify gene expression because of its accuracy and high sensitivity. The mRNA is quantified by reverse transcription of the isolated RNA to cDNA followed by quantitative PCR (qPCR) of the cDNA15. Fluorescent reporter molecules are used to examine the production of amplification products during each cycle of the PCR reaction (Figure 2). RT-PCR can be performed as two individual reactions or as a single reaction and precludes the use of gel electrophoresis16. Due to its sensitivity, it requires a relatively small amount of sample and can detect the expression of genes from a single cell15. This technique is used to study various mutations and to detect DNA methylations, and also to confirm the results obtained from other techniques, such as microarray analysis and RNA sequencing. The expression level of the genes can be quantified relatively or absolutely. The inherent quantitative potential of PCR makes this a quantitative as well as a qualitative assay16. It is currently one of the most important techniques used in the fields of molecular medicine, microbiology, and biotechnology as a diagnostic tool for the quantification of RNA17.
Repeated cocaine exposure dysregulates BDNF expression and signaling in the mesocorticolimbic pathway of the adolescent rat
Published in The World Journal of Biological Psychiatry, 2019
Lucia Caffino, Giuseppe Giannotti, Giulia Messa, Francesca Mottarlini, Fabio Fumagalli
Samples for RNA analysis were taken from the same animals used for protein measurements. Total RNA was isolated by single-step guanidinium isothiocyanate/phenol extraction using PureZol RNA isolation reagent (Bio-Rad Laboratories, Segrate, Milan, Italy) according to the manufacturer’s instructions and quantified by spectrophotometric analysis. Following total RNA extraction, the samples were processed for real-time reverse transcription polymerase chain reaction (real time RT-PCR) to assess mRNA levels, as described previously (Giannotti et al. 2014). In brief, an aliquot of each sample was treated with DNase to avoid DNA contamination. RNA was analyzed by TaqMan qRT-PCR instrument (CFX384 real time system, Bio-Rad Laboratories) using the iScriptTM one-step RT-PCR kit for probes (Bio-Rad Laboratories). Samples were run in 384-well formats in triplicate as multiplexed reactions. Data were analyzed with the comparative threshold cycle (ΔΔCt) method using 36B4 as the reference gene. The primer efficiencies were experimentally set up for each couple of primers.
Diagnostic Utility of Quantitative Polymerase Chain Reaction versus Culture in Endophthalmitis and Uveitis
Published in Ocular Immunology and Inflammation, 2019
Harpal Singh Sandhu, Amir Hajrasouliha, Henry J. Kaplan, Wei Wang
Infectious endophthalmitis is a rapidly progressive and potentially catastrophic disease in which early diagnosis and treatment are key. Historically, the gold standard of diagnosis has been culture and antibiotic sensitivity. However, the yield of culture is notoriously variable and frequently low. Polymerase chain reaction (PCR) techniques can also be used to diagnose other ocular infectious diseases. For fastidious infectious agents that are difficult to grow in the laboratory, PCR can be more effective and less time-consuming than conventional microbiology for detecting pathogens from intraocular fluids. Additionally, quantitative or real-time PCR (q- or rtPCR) has made it possible to quantify the viral or bacterial load associated with infectious diseases in the eye. Although there have been a number of studies assessing qPCR, new techniques are continually emerging, potentially improving the efficacy and decreasing the cost of this technology for clinicians.