Immunofluorscence  53BP1 [H-300]

Get tips on using HphI NEB#R0158 to perform Restriction Enzymes HphI

Get tips on using HindIII restriction enzyme to perform Restriction Enzymes HindIII

Get tips on using HindIII NEB #R0104 to perform Restriction Enzymes HindIII

Get tips on using HDAC1 Polyclonal Antibody to perform Western blotting HDAC1

Get tips on using Human ShhN ELISA to perform ELISA Human - ShhN

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 have 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 efficacy of transduction and shRNA on its target site.

Get tips on using HpyCH4III NEB#R0618 to perform Restriction Enzymes TaaI / HpyCH4III

Get tips on using HaeIII NEB#R0108 to perform Restriction Enzymes HaeIII / BsuRI

Get tips on using HpaII NEB#R0171 to perform Restriction Enzymes HpaII / MspI

Get tips on using Hoechst 33258 to perform DNA quantification Human - MDA-MB-231