China
Africa
Explore chapters and articles related to this topic
Approaches to the Measurement of Biological Pollutants
Published in Somenath Mitra, Pradyot Patnaik, Barbara B. Kebbekus, Environmental Chemical Analysis, 2018
Somenath Mitra, Pradyot Patnaik, Barbara B. Kebbekus
The methods of RNA isolation depends upon the tissue and the type of RNA to be extracted. Procedures to isolate total cellular RNA include chemical extractions and centrifugation. Phenol extraction was one of the first techniques to successfully isolate RNA from many sources. However, guanidinium salts have been found to be better options, even for those tissues that are rich in RNA degrading enzymes. Guanidinium hydrochloride and guanidinium thiocyanates are very powerful chaotropic agents. Guanidinium thiocyanate-based methods are quite popular for the isolation of good-quality RNA from a variety of tissues. Cells (or tissues) are homogenized directly in a solution containing guanidinium salt and reducing agents such as 2-mercaptoethanol (2-ME) or dithiothreitol (DTT) to break intramolecular protein disulfide bonds. These conditions rapidly inactivate RNases by distorting the secondary and tertiary folding of the enzymes when the cells are disrupted. Using these reagents, it is possible to isolate intact RNA even from RNase-rich tissues and cells.
Potentials of Polyhydroxyalkanoates for Repair of Skin Defects
Published in Tatiana G. Volova, Yuri S. Vinnik, Ekaterina I. Shishatskaya, Nadejda M. Markelova, Gennady E. Zaikov, Natural-Based Polymers for Biomedical Applications, 2017
Tatiana G. Volova, Yuri S. Vinnik, Ekaterina I. Shishatskaya, Nadejda M. Markelova, Gennady E. Zaikov
To get insight into the mechanism of the wound healing process, we detected the factors that characterized the degree of inflammatory process, vascularization, and formation of the new connective tissue at the defect site. Real-time RT-PCR was performed in the region of quantification of gene expression to the factors of rat angiogenesis (VEGF), inflammation (TNF-a), collagen type I (Col-1) (an indicator of connective tissue formation), keratin 10 (K10), and keratin 14 (K14) (indicators of the formation of the spinous and granulosa layers of the epidermis during regeneration) in order to quantify the level of the transcription of the genes responsible for inflammation, vascularization, and connective tissue formation. Total RNA was isolated from the granulation tissue of the treatment and control groups by extraction with a guanidinium thiocyanate-phenol-chloroform mixture from the RNA-Extran reagent kit. Then, from the RNA template, using reverse transcriptase, we synthesized cDNA with several types of oligonucleotide primers: with a mixture of random primers – hexa primers – and oligo-dT primer, following the procedure recommended by the manufacturer of Sintol. Negative controls of reverse transcription reaction were prepared for all samples, to confirm the absence of DNA contamination in the initial RNA. Real-time PCR-amplification for quantification of cDNA fragments of rat VEGF, rat TNF-a, rat Col-1, rat K10, and rat K14 was performed by using a CFX-96 thermocycler, according to the manufacturer’s protocol, using the color channel of HEX.
Elucidating the effect of different amino-functionalized spherical mesoporous silica characteristics on ribonucleic acid selectivity and adsorption capacity
Published in Journal of Asian Ceramic Societies, 2018
Ryouichi Hikosaka, Fukue Nagata, Masahiro Tomita, Katsuya Kato
Recently, ribonucleic acid (RNA) has been studied to elucidate the function of cells [1,2] and it has been applied in biosensors [3,4] and targeting therapies [5,6]. For such studies, the extraction and purification of RNA from cells is required. There are currently two commercially available procedures for purifying and extracting RNA that are very popular; the oligo thymidylic acid (oligo(dT))-modified cellulose column [7,8] and acid guanidinium thiocyanate–phenol–chloroform methods [8,9]. The former method, reported by Aviv and Leder [7], is used for the extraction of poly adenyl(A)-rich RNA, namely, messenger RNA (mRNA). The mRNA binds onto the column using the complimentary binding between the poly(A) tail of the mRNA and the oligo(dT) sequence on the column, and it is eluted by a buffer solution of a low ionic strength. This has the advantage of extracting gene-specific mRNA from cells. The latter method is a procedure combining the protein degradation caused by guanidinium thiocyanate and RNA isolation using phenol–chloroform. The method can effectively extract total RNA from cells. However, these methods have some problems, notably they are expensive, have complicated procedures, and can influence residual solvents during RNA applications.