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Trends in Cancer Screening: Different Diagnostic Approaches
Published in Anjana Pandey, Saumya Srivastava, Recent Advances in Cancer Diagnostics and Therapy, 2022
Anjana Pandey, Saumya Srivastava
However, miRNAs are such easy targets to quantify by PCR methods due to several reasons: (i) the small size of miRNA interferes with the optimal primer designing (ii) presence of stable hairpin structure in the pre-miRNA, (iii) omnipresence of different family members of miRNA that differ in only one or a few nucleotides, (iv) and most important, their low abundance in body fluids and interference in detection due to genomic contamination. Hence, genomic DNA contamination removal is required before the reverse transcription analysis. Furthermore, miniature miRNAs groups can be assessed with customized plates (e.g., Thermo Fisher) that are commercially available. Two main approaches combining the retro transcription and amplification steps are presently adopted to detect miRNA from qPCR. The first method ((Qiagen, Exiqon) comprises the use of poly(A) extension for cDNA synthesis before the reverse transcription process in a single step, disabling the restriction of short primer designing in qPCR. The assay is performed with double-stranded DNA binding dyes or SYBR green. In the second method (TaqMan technology), specific stem-loop primers are used to synthesize cDNA to enhance amplification specificity. Afterward, qPCR is performed by the fluorescent FAM method. RT-qPCR method of Exiquon is considered a more sensitive and specific miRNA detection method than the TaqMan Technology (Chugh and Dittmer, 2012; Leshkowitz et al., 2013; Ruiz-Tagle et al., 2020).
Functional DNa Building Blocks and Their Hydrogel Scaffolds for Biomedical Applications
Published in Gilson Khang, Handbook of Intelligent Scaffolds for Tissue Engineering and Regenerative Medicine, 2017
Seung Won Shin, Kyung Soo Park, Soong Ho Um
For diagnostic application of DNA hydrogels, target nucleotides have been shown to be detectable along with the designed DNA building block species. Dan Luo and his colleagues have manufactured a new multimodular, specific DNA nanostructure composed of different monomers. In this system, different Y-DNAs, which plug-in with the core of the DNA block, have multiple color codes of different quantum dots, photoresponsive PEGA moieties, and single oligos for target DNA detection (Fig. 7.3b).29 Engaged with targeted and complimentary sequences, the nanostructures can form dimers. The monomers in the dimer can polymerize via a photocrosslinkable process, resulting in a hydrogel. During the polymerization process, multiple color-coded signals, which originated from the quantum dots, are highly amplified (Fig. 7.3b). In addition to the provided oligonucleotides, heavy metal atoms (e.g., mercury (Hg2+)) can be monitored in situ and then removed from solution.46 Two successive tyrosine sequences interact more favorably with the existing Hg2+ compared to the Watson–Crick base pairs of adenosine and tyrosine. This enables the detection of Hg2+ (Fig. 7.3c). The existence of Hg2+ promotes secondary folding in single DNA strands. When the SYBR green I dye specifically binds to the double strands of DNA sequences, the formation of a secondary structure induces a strong green fluorescence emission. Such a strategy may make it possible to remove heavy metal compounds, such as mercury, and isolate very pure aquatic environments.
Experimental Protocols for Generation and Evaluation of Articular Cartilage
Published in Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi, Articular Cartilage, 2017
Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi
Quantitative reverse transcription PCR (qRT-PCR) allows for detection of relative amounts of mRNA for genes of interest compared with an internal housekeeping gene or control, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin, or 18S ribosomal RNA. The concept of qRT-PCR is the same as regular PCR, except the amount of PCR product amplified during each cycle is measured quantitatively. This is termed real-time PCR, which is often also somewhat confusingly abbreviated as RT-PCR or rtPCR. This allows for detection of relative amplification differences during the linear phase of PCR amplification. This detection and quantitative measurement is made using a fluorescent dye that either (1) binds to double-stranded DNA (SYBR Green) or (2) binds to a specific region in your gene of interest between the forward and reverse primers (probe). There are benefits and drawbacks to using either SYBR Green or a probe. SYBR Green is typically less expensive than probes and can work for all gene amplifications. On the other hand, SYBR Green binds to double-stranded DNA nonspecifically, so DNA product due to unwanted reactions will also be measured. Probes are sequence specific, and, therefore, ameliorate detection of off-target reactions and have the advantage that they can be designed with various fluorophores such that multiple genes can be amplified in the same reaction tube (multiplexing). However, probes are more expensive and more time-consuming to design, and using probes to multiplex requires significant optimization. The particular type of detection technique used will depend on the available primers for the gene of interest.
Cholesteric liquid-crystalline DNA – a new type of chemical detector of ionizing radiation
Published in Liquid Crystals, 2022
Maria A. Kolyvanova, Mikhail A. Klimovich, Anna V. Shibaeva, Ekaterina D. Koshevaya, Yuriy A. Bushmanov, Alexandr V. Belousov, Vladimir A. Kuzmin, Vladimir N. Morozov
Visualisation of the DNA CLCD samples was performed using a Leica TCS SP5 confocal laser-scanning microscope with LAS AF software (Leica Microsystem GmbH, Germany). A DNA-specific fluorescent dye SYBR Green I (SG; Lumiprobe, USA) was used as the fluorescent probe. The unbound SG has a low quantum yield of fluorescence (≈10–4); binding to DNA sharply increases this parameter (>1000-fold) [27]. This dye also does not fluoresce in a PEG solution. Staining of the samples was performed after radiation exposure to prevent degradation of the dye. A 50 µL drop of DNA CLCD sample stained with 1.44 × 10–6 M of SG was placed on a glass slide and covered with the cover slip. An Ar 488 nm laser was used for excitation the dye; the fluorescence was detected in the wavelength range of 500–600 nm. The analysis of particle size was performed using ImageJ software (National Institutes of Health, USA). At least 300 DNA CLCD particles were processed in each case.
Effects of life-stage and passive tobacco smoke exposure on pulmonary innate immunity and influenza infection in mice
Published in Journal of Toxicology and Environmental Health, Part A, 2022
Lei Wang, Maya Rajavel, Ching-Wen Wu, Chuanzhen Zhang, Morgan Poindexter, Ciara Fulgar, Tiffany Mar, Jasmine Singh, Jaspreet K. Dhillon, Jingjing Zhang, Yinyu Yuan, Radek Abarca, Wei Li, Kent E. Pinkerton
Nine neonate mice and 6 adult mice were randomly selected from each treatment group for real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. The ratio of males to females was 1:2 for neonates and 1:1 for adults. Total ribonucleic acid (RNA) was extracted from the spleen using an RNeasy kit with on-column DNase digestion (Qiagen, Valencia, CA) per the manufacturer’s instructions. Reverse transcription of 1 µg DNA-free total RNA was carried out with 4 units of Omniscript Reverse Transcriptase (Qiagen, Valencia, CA), and 1 μM of oligo(dT)15 primer in a final volume of 20 µl. Quantification of messenger RNA (mRNA) levels was done using the LightCycler System (Roche Diagnostics, Mannheim, Germany) with the QuantiTect SYBR Green PCR Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The primers for each gene were selected from published sources [IFN-a (Riffault et al. 2000), IFN-β (Roth-Cross et al. 2007), IFN-γ (Maffei et al. 2004), 18S (Roth-Cross et al. 2007)] and detailed in Table 2, herein. Data were analyzed with LightCycler software and quantified using the comparative threshold cycle (Ct) method (Pfaffl 2001) with mouse 18S as a reference gene to serve as an internal control.
Degradation of antibiotic resistance contaminants in wastewater by atmospheric cold plasma: kinetics and mechanisms
Published in Environmental Technology, 2021
Xinyu Liao, Donghong Liu, Shiguo Chen, Xingqian Ye, Tian Ding
SYBR Green I (Solarbio Science & Technology Co., Ltd., Beijing, China) is a fluorescent probe used to detect double strand (ds) DNA. SYBR Green I bond to the minor groove of ds DNA and emits green fluorescence. The staining protocol was conducted according to Han et al. [30]. Untreated and treated E. coli suspensions were mixed with lysozyme (100 μg/ml) and incubated at 37°C for 1 h to break the cell envelope and release the intracellular DNA. The above mixtures were then stained with SYBR Green I at a ratio of 1–20, 000 at 37°C for 15 min. A Fluoroskan™ FL Microplate Fluorometer (Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.) was applied for estimation of the green fluorescent intensity at an emission wavelength of 525 nm and an excitation wavelength at 485 nm.