China 
Africa 
Explore chapters and articles related to this topic
Molecular Genetic Approaches to Obesity
Published in Claude Bouchard, The Genetics of Obesity, 2020
Streamson C. Chua, Rudolph L. Leibel
The arguments described above which favor the brain (and specific regions within) as an organ in which genes critical to energy balance are expressed suggest another strategy for the identification and mapping of genes relevant to human obesity. cDNA libraries can be prepared from mRNA isolated from human and/or rodent brain (or specific regions). If desired, such libraries can be enriched for brain-specific clones by “substraction” of clones which are also expressed in other organs (e.g., liver). Using hybrid cell lines, irradiation-reduced hybrids, chromosomes-specific libraries, and YACs, cDNAs can be selected which map to regions of the mouse or human genome known by classical genetic studies to contain genes which affect energy balance. Large genetic crosses segregating for specific obesity-related mutations (e.g., ob or db) can be used to map such candidate genes relative to the desired mutation. Any clone which is nonrecombinant with the mutation in a sufficiently large number of meioses is potentially the gene itself. This approach is useful in efforts to clone mutations in unknown genes by positional techniques, but also generates an increasingly high-resolution map of organ-specific expressed sequences in a particular region of the genome. Such transcriptional maps of the genome will be extremely useful in facilitating the application of molecular genetic information to the study of integrated physiology.51
Tree pollen allergens
Published in Richard F. Lockey, Dennis K. Ledford, Allergens and Allergen Immunotherapy, 2020
Rosa Codina, Fernando Pineda, Ricardo Palacios
In principle, there are two strategies that can be applied to obtain cDNAs coding for allergens [38]. The first approach uses patients’ specific IgE for the isolation of allergen-encoding cDNAs from expression cDNA libraries that have been constructed from the allergen source (Figure 10.3). For this approach, mRNA is isolated from the allergen source and converted into a cDNA by reverse transcription. Subsequently, this cDNA is inserted into a vector (usually a phage vector) suitable for construction of an expression cDNA library. After infection of appropriate host cells (usually Escherichia coli bacteria), clones expressing allergens can be located with patient's serum-specific IgE using immune-screening technology. DNA from the positive clones is then isolated, purified, and subjected to sequence analysis. The second approach for the isolation of allergen-encoding cDNAs involves DNA-based screening technologies (e.g., DNA-based screening of libraries, polymerase chain reaction [PCR], or reverse transcriptase-PCR [RT-PCR] strategies). Once allergen-encoding cDNAs have been obtained using either approach, they can be inserted into expression vectors, and highly purified recombinant allergens can be produced in large amounts (Figure 10.3).
Steroid Hormone Receptors Involved in Reproduction: Mechanism of Action
Published in Robert E. Garfield, Thomas N. Tabb, Control of Uterine Contractility, 2019
Paul Robel, Baulieu Etienne-Emile
The clones thus obtained can, in turn, be used for the screening of the same or other cDNA libraries. As the steroid hormone receptors are large proteins, it has generally been necessary to orientate overlapping clones, in order to get the complete reading frame of the protein. Thereafter, cDNAs corresponding to highly conserved regions of the receptor allowed cloning, in other species, of the receptor of the same steroid hormone and even, in the case of the androgen receptor, to hybridize the appropriate library with a cDNA of glucocorticosteroid receptor, under less stringent conditions.
Exploitation of the antifungal and antibiofilm activities of plumbagin against Cryptococcus neoformans
Published in Biofouling, 2022
Weidong Qian, Wenjing Wang, Jianing Zhang, Yuting Fu, Qiming Liu, Xinchen Li, Ting Wang, Qian Zhang
cDNA library preparation and sequencing were conducted by Novogene Bioinformatics Technology Co., Ltd (Novogene, Tianjin, China). Total RNA samples for RNA-seq were extracted respectively from biofilm and planktonic cells in triplicate and prepared as described above using RNAiso Plus (Takara, Dalian, China) according to the manufacturer’s instructions. The RNA concentration and quality were examined using a NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE, USA) and Agilent 2100 bioanalyzer (Agilent Technologies, Waldbronn, Germany) . cDNA libraries were constructed using the NEBNext RUltraTM RNA Library Prep Kit (NEB, Ipswich, MA,USA), according to the manufacturer’s recommendations, and quantified using the Agilent 2100 bioanalyzer. qRT-PCR analysis was performed to determine effective concentrations. Then, cDNA libraries were sequenced using a Novoseq sequencer (Illumina, San Diego, CA, USA) to obtain 150 bp paired-end reads.
Development of neoantigens: from identification in cancer cells to application in cancer vaccines
Published in Expert Review of Vaccines, 2022
Nasim Ebrahimi, Maryam Akbari, Masoud Ghanaatian, Parichehr Roozbahani moghaddam, Samaneh Adelian, Marziyeh Borjian Boroujeni, Elnaz Yazdani, Amirhossein Ahmadi, Michael R. Hamblin
In classical approaches, majority of the neoantigens have been identified by screening of cDNA libraries. cDNA library screening is used to screen Tcell–reactive neoepitopes. In this method, the cDNA library and the relevant MHC molecules are up-regulated in cell lines, which are then co-cultured with T cells to identify the antigens that could induce T cell activation, as measured by cytokine secretion. Although the classical approach has led to the discovery of several neoantigens, it has low-throughput and is labor-intensive. CDK4 [46] and Multiple Myeloma 1 (MUM1) [47] were identified as the first neoantigens in melanoma using a cDNA library screening approach in 1995. Since melanoma tumors have high mutation rate, most of the neoantigens discovered by classical methods were in melanomas compared to other cancer types. However, some other mutations, such as HLA-A2 in renal cancer [48], and the EF2 mutation in lung cancer [49], were also detected by the cDNA library screening method.
High-efficiency unassisted transfection of platelets with naked double-stranded miRNAs modulates signal-activated translation and platelet function
Published in Platelets, 2021
Sophia Lazar, Jeremy G.T. Wurtzel, Xi Chen, Peisong Ma, Lawrence E. Goldfinger
RNA was extracted using TRIzol reagent according to the manufacturer’s instructions, except that 95% EtOH was used for RNA washes to preserve small RNAs. cDNA libraries were constructed using the NCode miRNA First-Strand cDNA Synthesis Kit (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instructions. Conventional PCR was carried out on an Eppendorf thermal cycler. Quantitative real-time PCR (qRT-PCR) analysis was performed using the LightCycler (Roche, Indianapolis, IN, USA) and the FastStart DNA Master SYBR Green I Kit, according to the manufacturer’s instructions. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a housekeeping control for qRT-PCR experiments for mRNAs, and hsa-miR-24-5p-1 was used as a housekeeping control for miRNAs. Primers used for conventional PCR and qRT-PCR amplification included the following: CD45-F: 5ʹ-ACAGCCAGCACCTTTCCTAC; CD45-R: 5ʹ-GTGCAGGTAAGGCAGCAGA; miR-223-3p-F: 5ʹ-TGTCAGTTTGTCAAATACCCA; Cel-miR-67-F: 5ʹ-TCACAACCTCCTAGAAAGAG; SEPT2-F: 5ʹ-TAAACAGCCTATTCCTAACT; SEPT2-R: 5ʹ- CCCCTCGCTCTTCAATTTC; P2RY12-F: 5ʹ-GAAGACCACCAGGCCATTTAAAAC; P2RY12-R: 5ʹ-GCCTGTTGGTCAGAATCATGTTAG; universal 3ʹ reverse primer from the NCode cDNA kit; GAPDH-F: 5ʹ-CATGGCCTTCCGTGTTCCTA; GAPDH-R: 5′-CCTGCTTCACCACCTTCTTGAT; hsa-miR-24-5p-1-F: 5ʹ-TGCCTACTGAGCTGATATCAGT. Single bands of predicted product size for each PCR reaction were confirmed by gel electrophoresis from all qRT-PCR samples. Gene expression levels relative to controls were determined using the 2−∆∆Ct method with housekeeping genes.