crispr-mouse-deletion-raw-264-7-dcstamp

Get tips on using Cell Counting Kit-8 to perform Cell cytotoxicity / Proliferation assay cell type - SMMC-7721, Huh7, Hep3B, 293T

Get tips on using jetPEI® DNA transfection, HTS application to perform DNA transfection Mammalian cells - Immortalized cell lines SMMC-7721

Get tips on using Cell Proliferation Reagent WST-1 to perform Cell cytotoxicity / Proliferation assay cell type - SMMC-7721, Huh7, Hep3B, 293T

Get tips on using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003 to perform ChIP Human - SMMC-7721

Get tips on using RNAqueous®-Micro Total RNA Isolation Kit to perform RNA isolation / purification Cells - immortalized ZR-75-1

Get tips on using QuikChange Site-Directed Mutagenesis Kit, 10 Rxn to perform Site Directed Mutagenesis (SDM) Human - Point mutation 786-O SIRT1

Get tips on using RNeasy Mini Kit to perform RNA isolation / purification Cells - Cancer cell lines Liver cancer cell lines Hepato cellular carcenoma (SMMC-7721, Huh7 & HepG2))

I would like to regulate the expression of a gene and in order to do that, I have purchased specific siRNA. After optimizing my transfection protocol and using electroporation I have achieved a 60-70% reduction of the gene of interest. However, I cannot observe a significant reduction of mRNA expression but only a reduction of protein. What might be the problem? Could the problem be in my cell treatment method?

Cell cytotoxicity assays measure the ability of certain compounds or chemical mediators to reduce the viability of the cells. The term cell cytotoxicity assay can sometimes be used interchangeably with cell proliferation assay. Healthy living cells can be identified by the use of formazan dyes, protease biomarkers or by measuring ATP content. The formazan dyes are chromogenic products formed by the reduction of tetrazolium salts by dehydrogenases, such as lactate dehydrogenase (LDH) and reductases that are released during cell death. Common tetrazolium salts include INT, MTT, MTS and XTT. Cell cytotoxicity can also be measured by using the SRB and WST-1 assays. These assays can usually be used in a high-throughput fashion and can be quantitated by measuring absorbance, colorimetry or luminescence. All these assays require similar numbers of cell plating at the initiation, a time course of treatment with the cytotoxic agent and at least triplicates for each condition at every point of analysis. Cell shrinkage, plasma membrane blebbing, cell detachment, externalization of phosphatidylserine, nuclear condensation and ultimately DNA fragmentation are well-described features of apoptosis. The assays that rely on cell membrane integrity for their function, may not be able to quantify early apoptosis. Therefore, in order to distinguish early apoptotic vs. late apoptotic or necrotic cells, additional flow cytometry techniques can be used. A combination of Annexin V and PI (propidium iodide) can be used to distinguish early (Annexin V+/PI-) and late apoptotic (Annexin V+/PI+) cells. Sometimes, caspase assays are used in order to differentiate the stages of apoptosis.

Get tips on using Pan Ras Monoclonal Antibody (Ras10) to perform Western blotting Ras