siRNA / miRNA gene silencing Human ACC-MESO1

As autophagy is a multi-step process which includes not just the formation of autophagosomes, but most importantly, flux through the entire system, including the degradation upon fusion with lysosomes, which makes it quite challenging for detection. There are several methods for detection in mammalian cells, including immunoblotting analysis of LC3 and p62 and detection of autophagosome formation/maturation by fluorescence microscopy, Currently, there is no single “gold standard” for determining the autophagic activity that is applicable in every experimental context, hence it is recommended to go for the combined use of multiple methods to accurately assess the autophagic activity in any given biological setting.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been greatly used for studies on embryonic development and cell differentiation.iPSCs provide a stable source for either self-renewal or differentiation into suitable cells when cultured in a particular environment. Pluripotent cell culture was originally started by deriving cells from inner cell mass (ICM) from pre-implanted blastocysts, these were called embryonic stem cells. These cells after isolation can be grown on traditional extracellular matrices (like mouse embryonic fibroblasts, MEFs) or feeder-free culture systems. DMEM/F12 has been the most commonly used basal media in the culture of pluripotent cells. These cells are cultured at normal atmospheric oxygen levels, 21%, however, some studies have proposed that 4% oxygen tension may be better for hESC growth. Higher D-glucose concentration (4.2g/l) and osmolarity (320mOsm) that mimics the natural environment of embryonic tissue are optimal for the growth of hESCs. Supplements like N2 and/or B-27, in the presence of growth factors like bFGF, have been shown to increase pluripotency of these cells. bFGF, FGF2 and other ligands of receptor tyrosine kinases like IGF are also required or maintain self-renewal ability of these cells. TGF𝛃1, by its activation of SMAD2/3 signalling, also represses differentiation of iPSCs. Other compounds like ROCK inhibitors reduce blebbing and apoptosis in these cells to maintain their clonogenicity. However, an inhibitor for LIF (leukaemia inhibitory factor, which is one of the pluripotent genes) has an opposing effect. Therefore, it is important to understand the culture conditions and media composition that affect downstream signalling in hESCs or iPSCs that may lead to their differentiation.

Protein isolation is a technique that involves isolation and/ or purification of protein from cells or tissues via chromatography or electrophoresis. The major challenges in protein isolation include: 1. The concentration of proteins in cells is variable and tends to be small for some intracellular proteins. Unlike nucleic acids, proteins cannot be amplified. 2. Proteins are more unstable than nucleic acids. They are easily denatured under suboptimal temperature, pH or salt concentrations. 3. Finally, no generalized technique/protocol can be applied for protein isolation. Proteins may have different electrostatic (number of positively or negatively charged amino acids) or hydrophobic properties. Therefore, protein purification requires multiple steps depending on their charge (a negatively charged resin/column for positively charged proteins and vice-versa), dissolution (using detergents) and unlike in the case of DNA and RNA, instead of using salts, proteins should be isolated by isoelectric precipitation.

The RNA-guided CRISPR-Cas9 nuclease system has revolutionized the genome editing practices. For the most part, the Cas9-mediated genome editing is performed either via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, However, designing of specific sgRNAs and minimizing off-target cleavage mediated mutagenesis are the major challenges in CRISPR-Cas based genome editing. To circumvent these issues, we can take advantages of many available tools and approaches for sgRNA construction and delivery.

The RNA-guided CRISPR-Cas9 nuclease system has revolutionized the genome editing practices. For the most part, the Cas9-mediated genome editing is performed either via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, However, designing of specific sgRNAs and minimizing off-target cleavage mediated mutagenesis are the major challenges in CRISPR-Cas based genome editing. To circumvent these issues, we can take advantages of many available tools and approaches for sgRNA construction and delivery.

The RNA-guided CRISPR-Cas9 nuclease system has revolutionized the genome editing practices. For the most part, the Cas9-mediated genome editing is performed either via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, However, designing of specific sgRNAs and minimizing off-target cleavage mediated mutagenesis are the major challenges in CRISPR-Cas based genome editing. To circumvent these issues, we can take advantages of many available tools and approaches for sgRNA construction and delivery.

The RNA-guided CRISPR-Cas9 nuclease system has revolutionized the genome editing practices. For the most part, the Cas9-mediated genome editing is performed either via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, However, designing of specific sgRNAs and minimizing off-target cleavage mediated mutagenesis are the major challenges in CRISPR-Cas based genome editing. To circumvent these issues, we can take advantages of many available tools and approaches for sgRNA construction and delivery.

The RNA-guided CRISPR-Cas9 nuclease system has revolutionized the genome editing practices. For the most part, the Cas9-mediated genome editing is performed either via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, However, designing of specific sgRNAs and minimizing off-target cleavage mediated mutagenesis are the major challenges in CRISPR-Cas based genome editing. To circumvent these issues, we can take advantages of many available tools and approaches for sgRNA construction and delivery.

Get tips on using Quick Amp Labeling Kit-one color to perform RNA amplification & labeling Mammalian - miRNA, Human Endometrial Stromal cells Cyanine 3-pCp

Transfection is a powerful technique that enables the study of the function of genes and gene products in cells. Based on the nature of experiments, we may need a stable DNA transfection in cells for persistent gain-of-function or loss-of-function of the target gene. For stable transfection, integration of a DNA vector into the chromosome is crucial which requires selective screening and clonal isolation. By carefully selecting a viral delivery system and related reagents we can ensure safe and highly-efficient delivery of expression constructs for high-level constitutive or inducible expression in any mammalian cell type.