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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
The extraction of RNA from 2D monolayer cultures is much easier than that from 3D culture and tissues. Many common manufacturers’ protocols exist for extracting RNA from monolayer, and most follow a straightforward approach of applying a lysis buffer, commonly guanidine based, to cells, followed by scraping and column cleanup. TRI-reagent, TRIzol, or QIAzol-based lysis or separation buffers also work and allow for potential separation of RNA, DNA, and protein from the same sample. The following protocol is based on the Qiagen RNeasy column kit, although many others are available.
Impact of high dietary selenium on the selenoprotein transcriptome, selenoproteome, and selenometabolites in multiple species
Published in Gary Bañuelos, Zhi-Qing Lin, Dongli Liang, Xue-bin Yin, Selenium Research for Environment and Human Health: Perspectives, Technologies and Advancements, 2019
Total RNA was isolated with TRIzol Reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. The rat studies used GeneChip Rat Genome 230 2.0 arrays (Affymetrix), which contain over 31,000 probe sets that target transcripts representing over 28,700 rat genes. The turkey transcripts were analyzed by RNA-Seq, using Illumina HiSeq 2500 paired-end analysis at UW Madison, which generated 28-M reads per sample. These reads were aligned on Build 102 of the turkey genome assembly 5.0 at the University of Minnesota.
Metal ion-directed assembly of two mixed-ligand coordination polymers: treatment effect on acute kidney injury by reducing urine Nagl content
Published in Inorganic and Nano-Metal Chemistry, 2021
Shi-Kai Tang, Ting-Ting Yan, Yan Luo, Wei Zhang
In the acute kidney injury animal model, after compounds 1 and 2 treatment, the inflammatory response in the glomerular epithelial cells was evaluated by real-time RT-PCR, reflected as the relative expression levels of the jak and stat. All the conduction was performed in accordance with the manufactures’ instruments with a little modification. In short, the acute kidney injury animal model was constructed as above described. Then compounds 1 and 2 were injected into the animal at the condition of 5 mg/kg for indicated treatment. After that, the glomerular epithelial cells were harvested from the mice of different groups. The TRIzol reagent was used to extract the total RNA. After measuring the concentration of the total RNA, all the RNA was transcripted into cDNA with RNA reverse transcription kit. In the end, the relative expression level of the jak and stat in the glomerular epithelial cell was measured with RT-PCR. The results were calculated using 2−ΔΔCt method from triplicate preformation.
Critical-Size Alveolar Defect Treatment via TGF-ß3 and BMP-2 Releasing Hybrid Constructs
Published in Journal of Biomaterials Science, Polymer Edition, 2019
Aybüke Garipcan, Petek Korkusuz, Elif Bilgic, Halil M. Aydin, Eda Ozturk, Ilyas Inci, Asya Ozkizilcik, K. Kamile Ozturk, Erhan Piskin, Ibrahim Vargel
To investigate the effect of TGF-β3 + BMP-2 release from PLLA/PCL scaffolds on bone formation, expression levels of osteogenesis related genes, ALP, BSP, OC, RUNX2, ON (SPARC) were investigated by real time quantitative PCR (qPCR) . Tissue samples encompassing the defected area were collected at 21 days (n = 5 for each group). Tissue samples were frozen immediately following surgical excision and stored at -80˚C until the RNA extraction was performed. The total RNA was isolated using TRIzol Reagent (Invitrogen Life Technologies, USA) according to the manufacturer's instructions. RNA concentrations and purity were determined by Thermo NanoDrop2000 (Thermo-Fisher Scientific, USA), and sample integrity assessed by gel electrophoresis. 1 µg of total RNA was reverse-transcribed using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Switzerland), according to the manufacturer’s instructions. The real-time qPCR reaction was performed on a LightCycler® Carousel-Based System (Roche, Switzerland) with FastStart Universal Probe Master (Roche, Switzerland). The list of gene specific primers and Universal ProbeLibrary (UPL) probes (Roche Diagnostic, Switzerland) were given in Table 1. Each experiment was conducted in triplicate. Gene expressions were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. Relative quantification of gene expression as fold change was calculated using ΔΔCT method [50].
Transcriptome analysis reveals that yeast extract inhibits synthesis of prodigiosin by Serratia marcescens SDSPY-136
Published in Preparative Biochemistry & Biotechnology, 2023
Junqing Wang, Tingting Zhang, Yang Liu, Shanshan Wang, Zerun Li, Ping Sun, Hui Xu
After 12 h of cell growth, samples were harvested for transcriptome analysis. Three duplicates were used for the experimental group grown in yeast extract (TR1, TR2, and TR3) and the control group grown in peptone (CK1, CK2, and CK3). TRIzol reagent (Invitrogen Life Technologies, USA) was used to isolate total RNA. To assess the quality and integrity, a NanoDrop spectrophotometer (Thermo Scientific, USA) and a Bioanalyzer 2100 system (Agilent, USA) were applied. The Zymo-Seq RiboFree Total RNA Library Kit (Zymo Research, USA) was used to remove ribosomal RNA (rRNA) from total RNA. The first strand of cDNA was constructed using random oligonucleotides and SuperScript III (Thermo Scientific). Following that, second-strand cDNA synthesis was performed with DNA polymerase I and RNase H (Thermo Scientific). Exonuclease/polymerase operations transformed the remaining overhangs into blunt ends and the enzymes were eliminated. As preparation for hybridization, Illumina PE adapter oligonucleotides were then ligated after the 3′ ends of the DNA fragments and adenylated. AMPure XP technology was employed to purify the library fragments and choose cDNA fragments of the preferred 400–500 bp length (Beckman Coulter, USA). Using the Illumina PCR Primer Cocktail, DNA fragments containing ligated adaptor molecules from both ends were preferentially selected for the 15-cycle PCR reaction. The products were separated using the AMPure XP system and measured using the high-sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent). The sequencing library was then sequenced on NovaSeq 6000 platform (Illumina) by Shanghai Personal Biotechnology Cp. Ltd.