Protein Expression Prokaryotic cells E. coli

Gene silencing through the use of small interfering RNA (siRNA) has become a primary tool for identifying disease-causing genes. There are several aspects for preparing and delivering effective siRNA to knockdown a target gene. The length of siRNA should be 21–23nt long with G/C content 30–50%. If a validated siRNA sequence for your target gene is not available, use siRNA generated against the entire target gene ORF. Always work with two or three different siRNA constructs to get reliable results. If you are not sure how much siRNA to use for a given experiment, start with a transfection concentration of 10-50 nM and use siRNA-specific transfection reagent to ensure efficient siRNA delivery in a wide range of cells.

Get tips on using LC3B Antibody to perform Autophagy assay cell type - Rat cerebral coritcal tissue

Get tips on using Fenozol to perform Stem cell Differentiation media Differentiation of Human iPSCs into Basal Forebrain cholinergic neurons (BFCN)

Get tips on using Click-iT™ TUNEL Alexa Fluor™ 488 Imaging Assay to perform TUNEL assay cell type - A549, NCI-H460, H1299 human alveolar carcinoma

Get tips on using BrainPhys™ Without Phenol Red to perform Stem cell Differentiation media Differentiation of Human iPSCs into Basal Forebrain cholinergic neurons (BFCN)

Get tips on using CD133 (Prominin-1) Monoclonal Antibody (13A4), APC, eBioscience™ to perform Immunohistochemistry Mouse - CD133

Get tips on using CD133 (Prominin-1) Monoclonal Antibody (13A4), APC, eBioscience™ to perform Flow cytometry Anti-bodies Human - CD133

Get tips on using Anti-Choline Acetyltransferase Antibody to perform Immunohistochemistry Mouse - ChAT

Get tips on using Anti-Choline Acetyltransferase Antibody AB5042 to perform Immunohistochemistry Mouse - ChAT

RNA quantification for appropriate concentration and quality (260/280 ratio) is an important step before downstream analysis (including sequencing, RT-qPCR, etc.). Having insufficient RNA quantities or a high salt or phenol in the RNA product can lead to variable or irreproducible downstream results. The various methods used for RNA quantification include: 1. UV spectrophotometric (challenges include: low sensitivity, cannot distinguish between nucleic acid species), 2. Fluorescence-based (challenges include: requires standards, cannot measure amplifiability, not sequence-specific), and 3. RT-PCR (challenges include: requires standards, time-intensive, costly). In order to overcome these challenges, and also to ensure the proper quantification and quality control for RNA product, it is important to use at least two or more methods in order to discard any inconsistencies. Using standards for calibrations increases the sensitivity range for RNA detention (fluorescence- and RT-PCR-based methods). When using RT- PCR, it is important to choose correct primers, aligning to the desired site on the template and of appropriate product length, along with positive, negative and loading controls. It is also important to have at least two primer pairs in order to confirm results.