Site Directed Mutagenesis (SDM) Human Deletion MCF-7

Get tips on using Human Breast Cancer Susceptibility Protein 2 (BRCA2) ELISA Kit to perform ELISA Human - BRCA2

When extracting nucleic acids from cell cultures, thorough homogenization of cells via vortexing in lysis buffer is very necessary. Choose the best RNA isolation method keeping in mind the downstream applications, generally, column-based isolations result in clean and concentrated RNA samples. Downstream applications like sequencing and cDNA synthesis require high-quality RNA, always treat the samples with DNases and check their integrity by running a gel.

RNAi or RNA interference is a common method to suppress gene expression in vitro/in vivo by utilizing the inherent microRNA machinery, without introducing a total gene knockout. 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.

RNAi or RNA interference is a common method to suppress gene expression in vitro/in vivo by utilizing the inherent microRNA machinery, without introducing a total gene knockout. 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 the 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.

RNAi or RNA interference is a common method to suppress gene expression in vitro/in vivo by utilizing the inherent microRNA machinery, without introducing a total gene knockout. 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.

Get tips on using NAPSIN A (TMU-AD02) ANTI-HUMAN MOUSE IGG MOAB to perform Immunohistochemistry Human - Naspsin A

An alternative to culture-based cell death detection is an assessment of other cell viability indicators using fluorescent dyes, including membrane potential and membrane integrity. Live/Dead assays differentiates live and dead cells using membrane integrity as a proxy for cell viability and are based on a fluorescent staining procedure followed by detection using flow cytometry. However, samples preparation for such flow cytometry-based techniques could be challenging. Cell harvesting by trypsinization, mechanical or enzymatic cell disaggregation from tissues, extensive centrifugation steps, may all lead to preferential loss of apoptotic cells. To overcome this strictly follow manufacturers instruction of the detection kit.