Chapter title |
Short Hairpin RNA (shRNA): Design, Delivery, and Assessment of Gene Knockdown
|
---|---|
Chapter number | 10 |
Book title |
RNA Therapeutics
|
Published in |
Methods in molecular biology, September 2014
|
DOI | 10.1007/978-1-60761-657-3_10 |
Pubmed ID | |
Book ISBNs |
978-1-60761-656-6, 978-1-60761-657-3
|
Authors |
Chris B. Moore, Elizabeth H. Guthrie, Max Tze-Han Huang, Debra J. Taxman, Taxman, Debra J., Moore, Chris B., Guthrie, Elizabeth H., Huang, Max Tze-Han |
Abstract |
Shortly after the cellular mechanism of RNA interference (RNAi) was first described, scientists began using this powerful technique to study gene function. This included designing better methods for the successful delivery of small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) into mammalian cells. While the simplest method for RNAi is the cytosolic delivery of siRNA oligonucleotides, this technique is limited to cells capable of transfection and is primarily utilized during transient in vitro studies. The introduction of shRNA into mammalian cells through infection with viral vectors allows for stable integration of shRNA and long-term knockdown of the targeted gene; however, several challenges exist with the implementation of this technology. Here we describe some well-tested protocols which should increase the chances of successful design, delivery, and assessment of gene knockdown by shRNA. We provide suggestions for designing shRNA targets and controls, a protocol for sequencing through the secondary structure of the shRNA hairpin structure, and protocols for packaging and delivery of shRNA lentiviral particles. Using real-time PCR and functional assays we demonstrate the successful knockdown of ASC, an inflammatory adaptor molecule. These studies demonstrate the practicality of including two shRNAs with different efficacies of knockdown to provide an additional level of control and to verify dose dependency of functional effects. Along with the methods described here, as new techniques and algorithms are designed in the future, shRNA is likely to include further promising application and continue to be a critical component of gene discovery. |
X Demographics
Geographical breakdown
Country | Count | As % |
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Unknown | 1 | 100% |
Demographic breakdown
Type | Count | As % |
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Members of the public | 1 | 100% |
Mendeley readers
Geographical breakdown
Country | Count | As % |
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United Kingdom | 5 | <1% |
United States | 3 | <1% |
France | 1 | <1% |
Italy | 1 | <1% |
Brazil | 1 | <1% |
India | 1 | <1% |
Germany | 1 | <1% |
Canada | 1 | <1% |
Denmark | 1 | <1% |
Other | 6 | <1% |
Unknown | 1212 | 98% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Student > Bachelor | 294 | 24% |
Student > Ph. D. Student | 259 | 21% |
Student > Master | 158 | 13% |
Researcher | 90 | 7% |
Student > Doctoral Student | 60 | 5% |
Other | 83 | 7% |
Unknown | 289 | 23% |
Readers by discipline | Count | As % |
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Biochemistry, Genetics and Molecular Biology | 375 | 30% |
Agricultural and Biological Sciences | 252 | 20% |
Medicine and Dentistry | 89 | 7% |
Neuroscience | 55 | 4% |
Pharmacology, Toxicology and Pharmaceutical Science | 42 | 3% |
Other | 114 | 9% |
Unknown | 306 | 25% |