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Genetics at the Cell Level
Published in Carlos Simón, Carmen Rubio, Handbook of Genetic Diagnostic Technologies in Reproductive Medicine, 2022
Valentina Lorenzi, Roser Vento-Tormo
In these protocols, individual barcoded gel beads and cells are flowed into a microfluidics device (Guo et al., 2012) where a mixture of aqueous and oil flows allows for their compartmentalization into a single droplet. Each barcode is composed of three segments: a PCR handle to initiate the PCR reaction; a cell barcode that is unique to each bead; and a unique molecular identifier (UMI) which is different for each cellular mRNA molecule. Cell barcodes and UMIs are generated by randomly assembling nucleotides. UMIs provide a means to remove PCR artifacts during the step of RNA amplification. Once the beads and cells are trapped in the droplets, a lysis buffer breaks the cell membrane and releases the mRNAs, which come into contact with the barcodes and ligate to them. This results in every mRNA molecule present inside the droplet being labeled with the same cell barcode and a different UMI. It is thus possible to subsequently employ massive parallel sequencing without losing the information about the cell of origin of each mRNA (Figure 2.4, central panel).
Experimental models and measurements to study cardiovascular physiology
Published in Neil Herring, David J. Paterson, Levick's Introduction to Cardiovascular Physiology, 2018
Neil Herring, David J. Paterson
However, changes in the expression and localization of mRNA do not always translate into changes in the protein levels. Western blotting is perhaps the most commonly used method of quantitatively assessing protein expression (see Figure 19.9). Tissue samples are homogenized in lysis buffer with a mixture of protease inhibitors, and the protein is isolated and denatured. The amount of protein isolated is then quantified and equal amounts from different samples are loaded onto a sodium dodecyl sulphate polyacrylamide gel and separated according to their molecular weight. The protein is then transferred (or blotted) onto a nitrocellulose or poly vinylidene difluoride membrane that can be blocked (to prevent background antibody binding) and incubated with a primary antibody against the protein of interest. After washing to remove any unbound primary antibody, the membrane is incubated with a secondary antibody (raised against the species of the primary antibody), which is linked to an enzyme such as horseradish peroxidase (which can cleave a chemiluminescent agent and subsequently be detected), or with a fluorescence label (which can be excited at one wavelength and its emission recorded at a different wavelength), as shown in Figure 19.10. The resulting chemiluminescence or fluorescence signal can be detected with a camera or photographic film and analysed with densitometry. However, the use of film is less than ideal because of the non-linearity of the image, especially if under- or overexposed, leading to inaccurate quantification.
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
Sperm cells are cast into miniature agarose gels on microscopic slides and lysed in situ to remove DNA-associated proteins in order to allow the compacted sperm DNA to relax. The lysis buffer (Tris 10 mmol/L, 0.5 mol/L EDTA, and 2.5 mol/L NaCl, pH 10) contains 1% Triton X-100, 40 mmol/L DTT, and 100 μg/mL proteinase K. The slide immersion time in alkaline lysis solution ranges between 1 and 20 minutes and does not affect assay results (210). Micro-gels are then electrophoresed (20 minutes at 25 V/0.01 A) in neutral buffer (Tris 10 mmol/L containing 0.08 mol/L boric acid and 0.5 mol/L EDTA, pH 8.2), during which time the damaged DNA migrates from the nucleus toward the anode. The DNA is visualized by staining the slides with the fluorescent DNA binding dye SYBR Green I. Comet measurements are performed manually or by computerized image analysis using fluorescence microscopy (Figure 6.2b) (150).
Discovery of dual S-RBD/NRP1-targeting peptides: structure-based virtual screening, synthesis, biological evaluation, and molecular dynamics simulation studies
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2023
Chunfang Hu, Ting Guo, Yunting Zou, Junyi Gao, Yi Gao, Miaomiao Niu, Yang Xia, Xiaozhou Shen, Jindong Li
To test the infection-blocking effect of peptides against SARS-CoV-2, pseudovirus affinity and infection experiments were conducted. The SARS-CoV-2 pseudovirus was derived from pseudotyped HIV-1 virus expressing S protein of SARS-CoV-2. To be detectable, the pseudovirus also contained a firefly luciferase gene. 293 T cells at a 2 × 104 density per well were seeded in 96-well plates and incubated overnight at 37 °C. The diluted ligands were preincubated with the pseudovirus for 1 h at 37 °C before being transferred to 293 T cells. Following 48 h of incubation, the cells were washed with 100 μl of PBS. 50 μl of lysis buffer was used to lyse cells. The luciferase detection reagent was included in this lysis buffer, and signals would be detected by using the luciferase detecting kit (according to instructions of manufacturer, Promega).
Potent activity of bioconjugated peptide and selenium nanoparticles against colorectal adenocarcinoma cells
Published in Drug Development and Industrial Pharmacy, 2019
V. R. Ranjitha, Umashankar Muddegowda, V. Ravishankar Rai
DNA fragmentation assay was carried out to detect the apoptosis in the treated cells as described previously by Saadat et al. [32]. Briefly, the cells seeded in 6-well plate were treated with a desired concentration of NP, peptide, and peptide-conjugated nanoparticle. The cells were harvested from the culture plate by discarding media. Overall, 500 µl of lysis buffer is added then incubated at 65 °C for 20 min and cooled to room temperature. Then 700 µl chloroform-isoamyl alcohol was added, and then centrifuged at 12,000 rpm for 5 min. The upper phase was transferred to a new Eppendorf tube and an equal volume of isopropanol was added and centrifuged at 12,000 rpm for 10 min. The pellet was air-dried and 50 µl of sterile water is added and electrophoresed at 1.5% agarose gel.
Dual-functionalized liposome by co-delivery of paclitaxel with sorafenib for synergistic antitumor efficacy and reversion of multidrug resistance
Published in Drug Delivery, 2019
Meng Lei, Guanglan Ma, Sijia Sha, Xueyuan Wang, Haiting Feng, Yongqiang Zhu, Xiao Du
To quantitative study intracellular uptake, MCF-7/MDR cells were seeded in 6-well plates at a density of 1 × 106 cells per well and cultured until a confluent monolayer of cell formed. Subsequently, different drug-loaded liposomes (PTX, 2 μg/mL) were added into each well and incubated with cells for 1, 2, 4 and 8 h, respectively. The original medium was then discarded and washed with PBS for three times. Thereafter, 150 μL of cell lysis buffer was added to fully lyse cells. The BCA Protein Assay Kit (Beyotime, China) was performed for determining the amount of protein, and the concentration of intracellular drug was detected by HPLC-MS-MS. The cellular uptake (Qdrug/Qprotein) was evaluated, where Qdrug and Qprotein represented the amount of drug and protein in MCF-7/MDR cells.