Abstract
Short hairpin RNA (shRNA) is a technique used to silence gene expression stably in various cells. There are however several reported problems. First, the cloning of oligos can lead to ligation of multiple copies; second, premature termination of sequencing reaction during confirmation of hairpin template; third, microdeletions/substitutions in hairpin during cloning; and fourth, off target effects. In this chapter, we have described a retrovirus transduction-based protocol that can be used on cells in culture without encountering any of the reported issues. We have used this protocol to clone shRNA templates for at least 10 different genes and confirmed them by dideoxy sequencing. The knockdown of 75-90% for two mRNA expressing genes, CDH5 and keratin KRT80, and a long non-coding RNA, XIST, is presented here.
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