Protein Expression Eukaryotic cells A. thaliana

Get tips on using DC™ Protein Assay Kit I to perform Protein quantification Tissue - mouse thymus

Get tips on using DC™ Protein Assay Kit I to perform Protein quantification Tissue - mouse spleen

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.

The RNA interference (RNAi) is used to inhibit gene expression or translation, by neutralizing targeted mRNA molecules. Two types of RNA molecules such as microRNA (miRNA) and small interfering RNA (siRNA) play a central role in RNAi. Few points have to considered to increase the transfection efficiency of siRNA. Always use healthy, actively dividing cells to maximize transfection efficiency. The confluency of cells should be between 50-70%. Always use the most appropriate siRNA concentration to avoid off-target effects and unwanted toxic side effects. Positive and negative controls should be used for each and every experiment to determine transfection efficiency.

Get tips on using AllPrep DNA/RNA/Protein Mini Kit to perform Protein isolation Tissue - Human placental tissue

Get tips on using IEF and 2-D Protein Standards to perform Protein Ladder IEF and 2-D Standards

Get tips on using Unstained Protein Ladder, Broad Range Ladder to perform Protein Ladder Unstained

Get tips on using SuperSignal™ Molecular Weight Protein Ladder to perform Protein Ladder Unstained

Get tips on using Pierce™ Unstained Protein MW Marker to perform Protein Ladder Unstained

Get tips on using Biotinylated Protein Ladder Detection Pack #7727 to perform Protein Ladder Immunofluorescence