Methods in Molecular Biology
Martin G. Pomper, Juri G. Gelovani, Benjamin Tsui, Kathleen Gabrielson, Richard Wahl, S. Sam Gambhir, Jeff Bulte, Raymond Gibson, William C. Eckelman in Molecular Imaging in Oncology, 2008
Real-time PCR, also called quantitative real-time PCR, is based on the same principle used in PCR, but it allows quantification of the target product after each round of amplification. In other words, instead of the qualitative nature of the PCR, the real-time PCR technique is quantitative. In this technique, commercially available fluorescence detecting thermocyclers are used to amplify specific nucleic acid sequences while simultaneously following the concentration of the products. There are two common methods of RT-PCR product quantification. One utilizes fluorescent dyes, such as SYBR green (asymmetrical cyanine dye), that intercalate with double-strand DNA. Another uses modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA as in Taqman technique (Fig. 6). This method involves the use of an oligonucleotide reporter probe where the fluorophore is quenched by another molecule. After annealing into the complementary DNA strand, the fluorophore inside the probe is still quenched. However, during PCR reaction, the probe is degraded by the 5′ → 3′ Taq polymerase activity and the reporter is then unquenched resulting in emission of fluorescence. The major advantages of realtime PCR are its high sensitivity and the ability to process many samples simultaneously.
Sperm chromatin assessment
David K. Gardner, Ariel Weissman, Colin M. Howles, Zeev Shoham in Textbook of Assisted Reproductive Techniques, 2017
The second electrophoresis is performed at 20 V (1 V/ cm), and 12 mA for four minutes in 0.03 mol/L NaOH. Then, the slides are rinsed in a neutralization buffer (0.4 mol/L Tris-HCl, pH 7.5) for five minutes, briefly washed in TBE buffer, dehydrated in increasing concentrations of ethanol, and air-dried. DNA is stained with SYBR Green I at a 1:3000 dilution in Vectashield® (Vector Laboratories, Burlingame, CA). Samples are assessed by visual scoring or digitalization and image processing. The frequency of sperm cells with fragmented DNA is established by measuring at least 500 sperm cells per slide. Cells are classified as undamaged or damaged based on the length of the tail, which contains DNA fragment single- stand breaks (up/down migration), DSBs (right/left migration), or both (207).
Japanese Encephalitis Virus and Human CNS Infection
Sunit K. Singh, Daniel Růžek in Neuroviral Infections, 2013
A sensitive quantitative assay for JEV RNA has been reportedly developed using real-time RT-PCR. The assay was performed using LightCycler and RNA amplification kit SYBR Green I by selecting the JEV specific primer from the 3’UTR. On comparing results obtained by real-time RT-PCR assay for JEV and infectivity titrations, it was suggested that the real-time RT-PCR assay could have an additive effect on the interpretation and evaluation of virus clearance, especially during the virus removal process (Jeong et al. 2003). Again, a one step TaqMan RT-PCR using a TaqMan probe has also been developed for detection of JEV, where it was shown to be 10-fold more sensitive than the conventional two-step RT-PCR method (Yang et al. 2004).
Effect of phosphate availability on biofilm formation in cooling towers
Published in Biofouling, 2020
Ingrid S. M. Pinel, Lan Hee Kim, Vitor R. Proença Borges, Nadia M. Farhat, Geert-Jan Witkamp, Mark C. M. van Loosdrecht, Johannes S. Vrouwenvelder
The methodology applied for determining the limiting nutrient was derived from Prest et al. (2016). Bacterial growth potential tests were performed in sterile containers. Recirculating water samples from the three CTs were collected onsite and immediately filtered through a 0.45 µm pore-size sterile nylon syringe filter (Sartorius) to avoid predation by higher organisms such as protozoa. Each sample was split into ten aliquots of 30 ml. The same nutrient compounds as dosed in the CTs were added to the aliquots as follows: no addition (‘Blank’), 1 mg-N + trace elements (‘N + M’), 1 mg-P (‘P’), 2 mg-C (‘C’), 2 mg-C + 1 mg-N + 1 mg-P + trace elements (‘All’). The test was performed in duplicate (n = 2). The aliquots were incubated at 37 °C in the dark with no shaking. Bacterial growth was monitored daily using a BD AccuriTM C6 flow cytometry (BD Biosciences) by staining with SYBR Green I (10,000 ×; Invitrogen). The final concentration of SYBR Green I in the samples was 1.96 µM. The staining protocol and flow cytometry analysis are described in the literature (Hammes et al. 2008; Prest et al. 2016). The incubation period was five days, after which the stationary phase was reached in all aliquots. The net growth was calculated by subtracting the cell count on day 0 from the cell count on day 5 for each growth test.
The bacterial siderophore enterobactin confers survival advantage to Salmonella in macrophages
Published in Gut Microbes, 2019
Piu Saha, Xia Xiao, Beng San Yeoh, Qiuyan Chen, Bhuvana Katkere, Girish Soorappa Kirimanjeswara, Matam Vijay-Kumar
Total RNA was isolated from unstimulated and stimulated BMDMs by using TRIzol reagent (Sigma) as described in the manufacturer’s protocol. mRNA was used to synthesize cDNA for qRT-PCR using SYBR green (Quanta) according to manufacturer’s protocol. qRT-PCR was performed in StepOnePlusTM real time PCR instrument (Life technologies). Sequence of primers used for qRT-PCR were (sense and antisense respectively): 36B4 5ʹ-TCCAGGCTTTGGGCATCA-3ʹ and 5ʹ-CTTTATTCAGCTGCACATCACTCAGA-3ʹ57; Fpn1 (ferroportin 1) 5ʹ-TTGTTGTTGTGGCAGGAGAA-3ʹ and 5ʹ-AGCTGGTCAATCCTTCTAATGG-3ʹ58; Hamp (hepcidin) 5ʹ-AGAAAGCAGGGCAGACATTG-3ʹ and 5ʹ-CACTGGGAATTGTTACAGCATT-3ʹ58; Dmt-1 (divalent metal transporter-1) 5ʹ GGCTTTCTTATGAGCATTGCCTA-3ʹ and 5ʹ-GGAGCACCCAGAGCAGCTTA-3ʹ.5936B4 was used to normalize the relative mRNA expression using the comparative Ct (2−ΔΔCt) method. Fold change was determined by comparison to the untreated cells.
Uncoupling proteins: are they involved in vitamin D3 protective effect against high-fat diet-induced cardiac apoptosis in rats?
Published in Archives of Physiology and Biochemistry, 2022
Zienab Alrefaie, Hossam Awad, Khadeejah Alsolami, Enas A. Hamed
The quantitative real-time polymerase chain reaction (qRT-PCR) was done using the KAPA SYBR® FAST qPCR Kit Master Mix (2X) Universal Cat no. KR0389. The experiment was done in duplicate for each sample in a 96 well plate which includes control samples, target genes, reference gene and non-template control for each gene PCR reactions were carried out using an ABI Step One Plus (Applied Biosystems) and started by initial denaturation at 95 °C for 10 min, followed by 34 cycles at 95 °C for 5 s, 65 °C for 10 s, and 72 °C for 15 s. Melting curve analysis was performed immediately after amplification by measuring the fluorescence of SYBR Green I during a temperature transition from 60 °C to 95 °C at 0.1 C/second. Fluorescence data are converted into melting peaks using the Light Cycler Software Step One Software v 2.1 (Applied Biosystems). Quantitative values were obtained from the threshold cycle (Ct) number; the Ct is the threshold cycle at which the fluorescence curve reaches an arbitrary threshold. The comparative cycle threshold (CT) method used was also called 2 (–ΔΔCT) to determine the mRNA relative expression. First, the ΔCT value for each sample was calculated by determining the difference between the CT mean of the target gene and the CT mean of the endogenous control gene such as GAPDH. Then, ΔΔCT was determined by calculating the differences of the ΔCT value of the calibrator samples and the test samples. The fold change or the mRNA expression level was calculated according to this formula: 2−ΔΔCT (Table 2).
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