Immunohistochemistry Collagen VII antibody [LH7.2] Mouse Human

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 MammoCult™ Human Medium Kit to perform 3D Cell Culture Media Human breast cancer MCF-7 cells-Mammospheres

Get tips on using ON-TARGETplus Human THBS2 siRNA to perform siRNA / miRNA gene silencing Human - Aortic smooth muscle cell TSP-2

Protein isolation is a technique that involves isolation and/ or purification of protein from cells or tissues via chromatography or electrophoresis. The major challenges in protein isolation include: 1. The concentration of proteins in cells is variable and tends to be small for some intracellular proteins. Unlike nucleic acids, proteins cannot be amplified. 2. Proteins are more unstable than nucleic acids. They are easily denatured under suboptimal temperature, pH or salt concentrations. 3. Finally, no generalized technique/protocol can be applied for protein isolation. Proteins may have different electrostatic (number of positively or negatively charged amino acids) or hydrophobic properties. Therefore, protein purification requires multiple steps depending on their charge (a negatively charged resin/column for positively charged proteins and vice-versa), dissolution (using detergents) and unlike in the case of DNA and RNA, instead of using salts, proteins should be isolated by isoelectric precipitation.

Get tips on using MammoCult™ Human Medium Kit to perform 3D Cell Culture Media Human breast cancer MDA-MB-231 cells-Mammospheres

Get tips on using Silencer® FANCD2 siRNA (human) to perform siRNA / miRNA gene silencing Human - 501 Mel and SK Mel 28 FANCD2

Get tips on using Human GDNF DuoSet ELISA to perform ELISA Rat - GDNF

Get tips on using ON-TARGETplus Human PPRC1 siRNA to perform siRNA / miRNA gene silencing Human - MCF-7 PRC (PGC-1α–related coactivator)/PPRC1

Get tips on using Human VEGF PicoKine™ ELISA Kit to perform ELISA Human - VEGF

Get tips on using Human Sonic Hedgehog ELISA Kit (ab100639) to perform ELISA Human - ShhN