rna-isolation-purification-tissue-mouse-lung

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.

DNA-protein interactions are studied by using ChIP. The basic steps in this technique are crosslinking, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. During ChIP, if chromatin is under-fragmented or fragments are too large which can lead to the increased background and lower resolution. Shorter cross-linking times (5-10 min) and/or lower formaldehyde concentrations (<1%) may improve shearing efficiency. If Chromatin is over-fragmented, then optimize shearing conditions for each cell type to improve ChIP efficiency. Over-sonication of chromatin may disrupt chromatin integrity and denature antibody epitopes. If you do not see any product or very little product in the input PCR reactions, add 5–10 μg chromatin per IP.

Get tips on using Mouse TNF alpha ELISA Kit (ab208348) to perform ELISA Mouse - TNF-alpha

Get tips on using Mouse TNF-alpha Quantikine ELISA Kit to perform ELISA Mouse - TNF-alpha

Get tips on using Mouse Lipocalin-2/NGAL DuoSet ELISA to perform ELISA Mouse - NGAL/LCN2

Get tips on using Mouse Dkk-1 Quantikine ELISA Kit to perform ELISA Mouse - Dkk-1

Short hairpin or small hairpin RNA (shRNA) is artificial RNA, which has a hairpin loop structure, and uses inherent microRNA (miRNA) machinery to silence target gene expression. This is called RNA interference (RNAi). These can be delivered via plasmids or viral/bacterial vectors. Challenges in shRNA-mediated gene silencing include 1. Off-target silencing, 2. Packaging shRNA encoding lentivirus, and 3. Stable transduction in cells. RNAi has been designed to have anywhere from 19-27 bs, but the most effective design has 19 bp. In case commercial shRNAs are not available, potential target sites can be chosen within exon, 5’- or 3’ UTR, depending on which splice variants of the gene are desired. One should use the latest algorithms and choose at least two different sequences, targeting different regions, in order to have confidence in overcoming off-target effects. A BLAST search after selecting potential design will eliminate potential off-target sequences. For the second challenge, sequencing the vector using primers for either strand (50-100 bp upstream) is suggested, along with using enzymatic digestion on agarose gel for the vector. Next, once the shRNA-containing vector is packaged in a virus, it is important to check the viral titer before transduction. Finally, using a marker in the lentiviral vector (fluorescent protein or antibiotic resistance), along with qPCR for target gene expression can help in determining the efficacy of transduction and shRNA on its target site.

Get tips on using Mouse ICAM1 ELISA Kit (CD54) (ab100688) to perform ELISA Mouse - ICAM-1/CD54

Get tips on using Mouse/Rat Osteopontin (OPN) Quantikine ELISA Kit to perform ELISA Mouse - OPN

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.