Bacterial cell culture media

The challenge in isolating RNA from S. aureus cells is the disruption of the cell wall. A lot of protocols employ enzymatic digestion (pretreatment) which may affect gene expression patterns of certain genes. Therefore physical disruption using beads is considered to be the better alternative.

Get tips on using Fenozol to perform Cell cytotoxicity / Proliferation assay cell type - HUVEC

Get tips on using ATP Assay to perform Cell cytotoxicity / Proliferation assay cell type - adipose stem cells

Get tips on using LC3B Antibody #2775 to perform Autophagy assay cell type - Mast cell

Get tips on using SPLInsert™ Hanging to perform Cell migration / Invasion cell type - HaCat

Get tips on using Dynabeads™ Untouched™ Human B Cells Kit to perform Cell Isolation B cell

Get tips on using 24-Well Haptotaxis Assays, Collagen I to perform Cell migration / Invasion cell type - HEK293T

Get tips on using MTT Assay solution to perform Cell cytotoxicity / Proliferation assay cell type - adipose stem cells

miRNA is the inherent gene silencing machinery which can have more than one mRNA target, whereas siRNA can be designed to target a particular mRNA target. By design, both siRNA and miRNA are 20-25 nucleotides in length. The target sequence for siRNAs is usually located within the open reading frame, between 50 and 100 nucleotides downstream of the start codon. There are two ways in which cells can be transfected with desired RNAi: 1. Direct transfection (with calcium phosphate co-precipitation or cationic lipid mediated transfection using lipofectamine or oligofectamine), and 2. Making RNAi lentiviral constructs (followed by transformation and transduction). Lentiviral constructs are time consuming, but provide a more permanent expression of RNAi in the cells, and consistent gene silencing. Direct transfection of oligonucleotides provides temporary genetic suppression. Traditional methods like calcium phosphate co-precipitation have challenges like low efficiency, poor reproducibility and cell toxicity. Whereas, cationic lipid-based transfection reagents are able to overcome these challenges, along with applicability to a large variety of eukaryotic cell lines. When using oligos, the ideal concentration lies between 10-50nM for effective transfection.

The biggest problem in isolating RNA from gram-positive bacteria is the disruption of the cell wall. A lot of protocols employ enzymatic digestion (pretreatment) which may affect gene expression patterns of certain genes. Therefore physical disruption using beads could be the best alternative.