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Investigation of DNA Methylation in Autosomal Dominant Polycystic Kidney Disease
Published in Jinghua Hu, Yong Yu, Polycystic Kidney Disease, 2019
Unlike working with blood and urine samples, using kidney tissue is advantageous in that a larger yield of genomic DNA can be obtained. However, sample collection is invasive and not easy to get. In mouse studies, an appropriate method is used to euthanize the mouse, followed by perfusion of the kidney to get rid of excess blood. Once the kidneys are harvested, the tissue sample should either be processed fresh or flash frozen and stored at −80°C until needed. When working with frozen tissue, tissue should be removed from the freezer and allowed to thaw on ice if necessary. With an appropriate method, such as the phenol/chloroform extraction technique, genomic DNA is extracted from the tissue, quantified, and either used immediately or stored at −20°C.
Molecular Methods for the Diagnosis of Fungal Infections
Published in Attila Lorincz, Nucleic Acid Testing for Human Disease, 2016
Adam M. Bressler, Christine J. Morrison
Although traditional in-house methods have been shown to provide the greatest DNA yields at the lowest cost, these benefits must be balanced by practicality. Methods that may be acceptable for research purposes or for retrospective analyses may be difficult to incorporate into general clinical practice. Therefore, the application of complex enzymatic disruption methods, particularly in conjunction with phenol–chloroform extraction procedures, may not be feasible in a clinical laboratory setting.
Molecular Diagnostics: Present and Future
Published in Johan A. Maertens, Kieren A. Marr, Diagnosis of Fungal Infections, 2007
Holger Hebart, Juergen Loeffler, Hermann Einsele
For fungal DNA release and purification, phenol-chloroform extraction procedures were successfully applied (22). However, these protocols were time-consuming and rely on toxic chemicals. Furthermore, the cationic detergent cetyl trimethyl amonium aromide (CTAB) was successfully used to release Aspergillus-ONA (23) from blood and sputum specimens. The use of commercially available kits shortened the duration of DNA extraction (24). Investigators should keep in mind that the sensitivity of these kits varies widely. In our experience, only the Qiamp Tissue Kit® (Qiagen, Hilden, Germany) showed a comparable sensitivity of 10 cells/mL blood to the in-house method, whereas the lowest sensitivity was seen with the DNAzol kit (1000 cells/mL blood). The commercial kits are more expensive than any of the in-house methods. In another study, different DNA extraction methods were comparedfor the extraction of fungal DNA from sera artificially spiked with genomic DNA from Candida species (25). DNA purity and the detection limit were superior for the QIAamp DNA blood kit (Qiagen) and boiling of sera in an alkaline guanidine-phenol-Tris reagent compared to proteinase ढ digestion followed by organic extraction, the HighPure PCR template kit (Roche, Mannheim, Germany) and DNAzol (Sigma, Deissenhofen, Germany).
Large-scale quantitative genomics analyzes the circRNA expression profile and identifies the key circRNA in regulating cell proliferation during the proliferation phase of rat LR
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2019
Xueqiang Guo, Wei Jin, Cuifang Chang, Yi Ding, Yahao Wang, Lifei Li, Yanhui Chen, Jingbo Zhang, Cunshuan Xu, Guangwen Chen, Jianlin Guo
To validate the data from high-throughput sequencing, the candidate key circRNAs were amplified with specific divergent primer in the PH groups and the control groups. Each pair divergent primers were designed according to the corresponding linear transcript sequence and synthesized in Sango Biotech (Shanghai, China). Specific primers designed for circRNAs are listed in Table 1. Total RNAs from regenerating liver and normal tissues were extracted using the TRIzol reagent (Invitrogen, USA) according to the manufacturer’s protocol. RNA quality was estimated by spectrophotometry and agarose gel electrophoresis. Then the total RNAs was incubated 1 h at 37 °C with 20 units of RNase R (Epicentre Biotechnologies). RNA was subsequently purified by phenol-chloroform extraction. 1 μg of RNA per sample was reverse transcribed into cDNA using a cDNA Reverse Transcription Kit (Takara, Tokyo, Japan). The PCR program was as follows: 95 °C for 1 min, followed by 40 cycles of 95 °C for 30 s, and 60 °C for 30 s and 72 °C for 45 s. Expression of circRNAs was normalized to GAPDH (internal standard control) and calculated using the 2−ΔΔCt method. All qRT-PCRs were performed in triplicate.
A comparison of the genetic and clinical risk factors for arterial hypertension between indigenous and non-indigenous people of the Shoria Mountain Region
Published in Clinical and Experimental Hypertension, 2018
Tatyana Mulerova, Michael Ogarkov, Evgenya Uchasova, Michael Voevoda, Olga Barbarash
Blood for biochemical analysis was taken from the cubital vein in the morning in a fasting state; it was then centrifuged, and the blood serum was frozen and stored at temperatures below freezing. The material was transported to the laboratory in liquid nitrogen-refrigerated containers to avoid thawing. Data related to total cholesterol, high-density lipoprotein cholesterol (HDL-C), triglycerides (TGs), and low-density lipoprotein cholesterol (LDL-C) in serum were evaluated using standard testing systems (Thermo Fisher Scientific Oy, Vantaa, Finland) in a Konelab 30i biochemistry analyzer (Thermo Fisher Scientific). High lipid level was assessed in accordance with the 2012 European guidelines. A plasma fasting glucose level of 5.6–6.9 mmol/L was regarded as a carbohydrate metabolism disorder (fasting hyperglycemia). DNA extraction from blood was performed using a phenol-chloroform extraction technique. Polymorphisms of genes ACE (I/D), ADRB1 (Ser49Gly, A/G, rs1801252), ADRA2B (I/D), MTHFR (C677T, Ala222Val, rs1801133), and eNOS (4b/4a) were assessed by PCR using the following techniques: Snapir et al.; Lima et al.; Salimi et al. (15–17).
Molecular-genetic diagnostics of von Hippel-Lindau syndrome (VHL) in Bulgaria: first complex mutation event in the VHL gene
Published in International Journal of Neuroscience, 2018
Maria Glushkova, Petia Dimova, Iglika Yordanova, Tihomir Todorov, Ivan Tourtourikov, Vanyo Mitev, Albena Todorova
The deletion and duplication screening along the VHL gene was performed by multiplex ligation-dependent probe amplification (MLPA) analysis with standard MLPA kit for the VHL gene (SALSA MLPA P016 VHL probemix), following the manufacturer's instructions [24]. The DNA samples were purified by standard phenol/chloroform extraction. Between 50 and 200 ng of DNAs were diluted in TE buffer up to 4 μl total volume. The diluted samples were subjected to a short denaturation for 10 min in 1 μl denaturing buffer supplied within the MLPA kit. The hybridization with the VHL-specific probes as well as a number of control probes was performed at 60 °C overnight. In the ligation reaction, the hybridized probes were ligated by a specific ligase provided by the manufacturer. The final step represents the PCR amplification of the obtained fragments. The PCR primers, the enzyme dilution buffer and the polymerase are provided in the kit.