BioTechniques最新文献

Secondary metabolites in mangroves often interfere with RNA extraction yielding poor concentration and quality, which is unsuitable for downstream applications. As existing protocols yielded low-quality RNA from root tissues of Kandelia candel (L.) Druce and Rhizophora mucronata Lam., an optimized method was developed for improving the quality and yield of RNA. Compared with three other methods, this optimized protocol gave better RNA yield and purity for both species. The absorbance ratios were ≥1.9 for A260/280 and A260/230, while RNA integrity number values ranged from 7.5 to 9.6. Results show that our modified method is efficient in obtaining high-quality RNA from mangrove roots and is suitable for downstream experiments such as cDNA synthesis, real-time quantitative PCR and next-generation sequencing.

We formulated a method to synthesize 1 kbp DNA fragments using 'oligomer unidirectional joining method' via asymmetric extension supported by a simulator for oligonucleotide extension (AESOE). In this study, trials were conducted on 41 sets of different genomic pieces of ten flaviviral genomes, and 31 bacterial 16s rRNA fragments with sizes ranging from 500 bases to 1.0 kbp. Synthetic gene production was found to be successful in all those sets. The synthesis method has three steps: the first step is a seven-linked AESOE, the second step is the linking of the 400-base fragments from the first step, and the third step is the final amplification. Our present approach is highly reproducible and may no longer require optimization of oligomer design.

For difficult overlap extension PCR, a Gibson assembly process was inserted between the two PCR rounds to facilitate the formation of complete gene templates at a moderate temperature. That is, after amplifying each DNA fragment, they were preluded by a Gibson assembly process in equal proportion. Then, the assembled mixture was used as a template for the second PCR round. This idea was tested and verified by taking the cloning example of a single and a double site mutation of the retinoblastoma gene. This scheme associates overlap extension PCR with Gibson assembly exquisitely, significantly improving gene amplification efficiency, particularly in the fusion of long genes and multifragments using overlap extension PCR.

Mesenchymal stem cells are frequently utilized in the study of regenerative medicine. Electric fields (EFs) influence many biological processes, such as cell proliferation, migration and differentiation. In the present study, a novel device capable of delivering a direct current of EF stimulation to cells cultured in vitro is described. This bioreactor was customized to simultaneously apply a direct-current EF to six individual cell culture wells, which reduces the amount of experimental time and minimizes cost. In testing the device, adipose-derived mesenchymal stem cells stimulated with an EF in the bioreactor exhibited a greater cell proliferation rate while retaining stemness. The results provide a unique perspective on adipose-derived mesenchymal stem cell proliferation, which is needed for tissue engineering and regenerative medicine.

[Formula: see text] With advances in imaging technologies and the development of artificial intelligence-based predictive software, has the protein folding problem finally been solved?

Designed donor DNA delivery through viral or nonviral systems to target loci in the host genome is a critical step for gene therapy. Adeno-associated virus and lentivirus are leading vehicles for in vivo and ex vivo delivery of therapeutic genes due to their high delivery and editing efficiency. Nonviral editing tools, such as CRISPR/Cas9, are getting more attention for gene modification. However, there are safety concerns; for example, tumorigenesis due to off-target effects and DNA rearrangement. Analysis tools to detect and characterize on-target and off-target genome modification post editing in the host genome are pivotal for evaluating the success and safety of gene therapy. We developed Target-seq combined with different analysis tools to detect the genome integration site, DNA translocation and off-target events.

Here, the authors report a simple method to perform antigen retrieval using a commonly available commercial Instant Pot^® for immunohistochemistry. It provides a validated alternative to previous antigen retrieval methods that employ water baths, microwave ovens or scientific-grade pressure cookers. The Instant Pot can be set to obtain a variety of desired temperatures and is straightforward to use, making it extremely amenable to optimization. The Instant Pot method is an easy, safe and inexpensive alternative means to perform immunohistochemistry on formalin-fixed paraffin-embedded sections. It has been validated using several different monoclonal antibodies including ones directed against cell surface or intracellular antigens. As a result, it should be useful for a variety of research labs as well as undergraduate laboratory courses.

High-purity total RNA extraction from animal embryos is essential for transcriptome analyses. lampreys, together with hagfish, are the only extant jawless vertebrates or cyclostomes and are thus key organisms for EvoDevo studies. However, extracting uncontaminated RNA from early-stage embryos remains challenging. RNA does not bind to the silica membrane in filter-based extractions, significantly reducing yields; and ethanol/isopropanol precipitation methods lead to contaminants, bringing down the optical density (OD) 260/280 ratio. The RNA extraction protocol was modified using precentrifugation and adding salts before isopropanol precipitation. This modification significantly increased RNA yield, removed contaminants and improved RNA integrity. Egg membrane sources were suspected to cause RNA purification problems because low-quality extraction does not occur in posthatching embryos.

High-quality RNA isolation from recalcitrant adipose tissue with high lipid content and low cell numbers is difficult. Many studies have made efforts to optimize methods for isolating RNA from adipose tissue through combinations of column-based kits and phenol-chloroform methods, or through in-house protocols. However, the considerable complexity of these protocols and the various kits/materials required hamper their wide use. Herein, we describe an optimized protocol based on TRIzol reagent, which is the most accessible ready-to-use reagent for nucleic acid and/or protein isolation in laboratories. This article provides a step-by-step protocol yielding sufficient and qualified RNA from lipid-rich specimens for downstream applications.