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Sperm chromatin assessment
Published in David K. Gardner, Ariel Weissman, Colin M. Howles, Zeev Shoham, Textbook of Assisted Reproductive Techniques, 2017
Ashok Agarwal, Rakesh Sharma, Gulfam Ahmad
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).
Effects of selenium on SePP and Apo B-100 Gene expressions in human primary hepatocytes
Published in Robert Hofstra, Noriyuki Koibuchi, Suthat Fucharoen, Advances in Biomolecular Medicine, 2017
M. Putri, N. Sutadipura, S. Achmad, C. Yamazaki, S. Kameo, H. Koyama, M.R.A.A. Syamsunarno, T. Iso, M. Kurabayashi
After incubation in various concentrations of sodium selenite (0, 25, 50, 100, and 200 nM) for 72 h, total RNA was isolated from Hc cells using TRIzol reagent (Invitrogen, CA, USA). RNA was prepared by reverse transcription using oligo-dT and dNTP, and each sample was processed with the RT-PCR kit (TAKARA, Japan). Quantitative real time-PCR was performed using the SYBR Green PCR Master Mix (Applied Biosystems, CA, USA) according to the manufacturer’s instructions, and then evaluated using the Light Cycler 480 Real-Time PCR system (Roche, CA, USA). The expression level of the target gene was normalized against GAPDH mRNA levels. The sequences of primers for quantitative real-time PCR used in this study are listed in Table 1.
Detection Techniques for Single Nucleotide Polymorphisms
Published in Attila Lorincz, Nucleic Acid Testing for Human Disease, 2016
W. Mathias Howell, Johan Stenberg, Chatarina Larsson, Mats Nilsson, Ulf Landegren
Following removal of excess PCR reagents and the non-biotinylated strand, a detection probe is hybridized over the polymorphic positions on the solid phase targets. The SYBR Green I dye is added in solution and allowed to bind to the DNA duplexes. The samples are heated through a temperature range while fluorescence is continually monitored. As the melting temperature (Tm) of each allele-specific duplex is surpassed, the DNA strands separate, releasing the dye and causing a dramatic drop in fluorescence. The DNA duplexes formed with targets containing the 100% complementary or “matched” allele are more stable than duplexes formed with the alternate or “mismatched” allele.
The expression of hepatoma upregulated protein in human endometrium during the menstrual cycle
Published in Gynecological Endocrinology, 2021
Akimasa Takahashi, Akiyoshi Yamanaka, Akie Takebayashi, Fuminori Kimura, Takashi Murakami
Total RNA was extracted from the samples and purified using TRIzol Reagent with the PureLink™ Mini Total RNA Purification System (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The PrimeScript RT Reagent Kit (Takara Bio, Shiga, Japan) was used to synthesize complementary DNA. PCR samples were prepared up to a final volume of 20 μL using 1× SYBR Green Master Mix (Roche, Laval, Canada). Primer sequences used were 5′-GAAAGCAGGAGCAGCATAGAAGA-3′ (sense) and 5′- CACCAGCAAGAAGAGGCAAAC-3′ (antisense) for HURP and 5′-AAATCCCATCACCATCTTCCA-3′ (sense) and 5′-AATGAGCCCCAGCCTTCTC-3′ The PCR cycling conditions were as follows: 95 °C for 5 min amplification, 40 cycles of 94 °C for 15 s, 60 °C for 45 s, and 72 °C for 10 s on the Roche LC480 system (Roche) Cycle threshold (Ct) values were used to compute levels of mRNA expression from the standard curve. Analytical data were adjusted based on the mRNA expression of GAPDH as an internal control.
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.
Loop-mediated isothermal amplification methods for diagnosis of visceral leishmaniasis (kala-azar) – a systematic review
Published in Expert Review of Molecular Diagnostics, 2020
Gilberto Silva Nunes Bezerra, Walter Lins Barbosa Júnior, Amanda Virgínia Batista Vieira, Amanda Tavares Xavier, Manoel Sebastião Da Costa Lima Júnior, Edeneide Maria Xavier, Elis Dionísio Da Silva, Nilma Cintra Leal, Zulma Maria De Medeiros
Based on the studies mentioned above, blood marrow and whole blood when compared to other clinical specimens were able to achieve higher rates of sensitivity and specificity according to Verma et al. [21], Verma et al. [17], Mukhtar et al. [16], Dixit et al. [13] and Avelar et al. [11], which satisfy WHO standard rates [33] and corroborate with the great analytical sensitivity of 1 fg shared for them. Heat block has been extensively applied for isothermal amplification assays due to several advantages such as shorter reaction time when compared with other molecular techniques and priceless when compared to a thermocycler [20]. Moreover, LAMP positive results by turbidity visualization and SYBR Green I color change were more common because they can eliminate the chances of contamination, they do not require carcinogenic agents as ethidium bromide and they can reduce cost and time of reaction [17,22].