Navigation Links
New method powerfully boosts efficiency of RNA interference (RNAi) in shutting down genes
Date:2/24/2011

Cold Spring Harbor, N.Y. A research team led by scientists at Cold Spring Harbor Laboratory (CSHL) has developed a powerful method that allows them to sift through thousands of candidate hairpin-shaped RNA molecules at a time and pull out only those RNAs that potently shut down the activity of a target gene. This accomplishment will now allow biologists to fully exploit RNA interference (RNAi), a natural cellular mechanism that has already been co-opted by scientists for myriad purposes such as hunting for cancer genes, stopping viral infections and more recently, treating diseases in clinical trials.

"RNAi is a powerful tool that in theory can be used to knock down any gene of interest but has been difficult to implement in practice," says CSHL Professor and Howard Hughes Medical Institute (HHMI) Investigator Scott Lowe, Ph.D., who co-led the team with Gregory Hannon, Ph.D., also a CSHL Professor and HHMI investigator. One major challenge has been the difficulty of finding the correct molecular trigger for RNAi. The trigger is a tiny piece of RNA, which, by attaching to a matching piece of the target gene's RNA, spurs its destruction, thereby shutting down the production of protein from that gene.

"For every gene, depending on the size of its protein-coding RNA, there are potentially 500 to 5000 different small RNAs that can trigger RNAi," explains Hannon. Most of these are weak triggers that do not shut down gene activity completely or end up targeting a different gene resulting in so-called "off-target" effects. Either scenario can ruin experiments that use RNAi to study diseases in the lab, or worse, lead to useless, or even toxic, treatments in the clinic. "Picking the right trigger has been like trying to pull a needle out of a haystack," Lowe says.

In a paper that appears online on February 24 in the journal Molecular Cell, scientists from the Lowe and Hannon laboratories and their collaborators, including Professor and HHMI investigator Stephen Elledge, Ph.D., of Harvard Medical School, explain how they succeeded in solving this problem. Based on an idea by Hannon, a pioneer in RNAi technology, and using molecular tools developed in the Lowe laboratory, the team designed an assay that tests thousands of short hairpin RNA (shRNA) molecules at a time for their ability to shut down genes of interest in cells and identifies the most potent RNAi triggers.

In a pilot experiment to find the most potent RNAi triggers for nine target genesincluding a few hard-to-suppress cancer genesthe team generated genetic codes for about 20,000 shRNAs, each with the potential to shut down any one of the nine target genes. Each genetic code was then inserted into a retrovirus that was also engineered to carry the target geneor the "sensor"and a gene for a fluorescent protein, called a "marker." This design ensured that when cells growing in dishes were infected with the genetically engineered viruses, both the sensor and the marker would always be co-produced within each cell along with one shRNA.

ShRNAs that were inefficient at triggering RNAi failed to spur the destruction of their target (or sensor) genes' RNA and that of the fluorescent marker. So these cells produced a fluorescent protein whose brightness could easily be detected. But shRNAs that were potent RNAi triggers caused the efficient destruction of the target gene's RNA and that of the fluorescent marker as well. Those host cells therefore lacked all traces of fluorescence.

"All we had to do then was to sort these cells out, pull out each cell's genetic material and sequence the short hairpin RNA," explains graduate student Christof Fellman, who together with post-doctoral fellow Johannes Zuber led these efforts. "This gave us the identity of the RNAi trigger that was most potent in shutting down the target gene."

The team found that for each of the nine genes, only about 2.5% of all possible shRNAs against that gene could shut down its activity efficiently. The success of their approach also means that scientists might no longer have to rely on current algorithms that are often not accurate in predicting which shRNA would work best at shutting down any given gene.

"There is still very little that is known about small RNA biogenesis," says Hannon. "But this assay has now allowed us to explore this process at a scale that wasn't feasible before." The scientists' analysis of the 20,000 small hairpin RNAs, and especially those recovered from cells in which the target gene had been efficiently shut off has revealed new insights into this process and drastically improved the recipe for creating a potent RNAi trigger. "Our assay has given us a strong framework for rational design of small hairpin RNAs," says Lowe.


'/>"/>

Contact: Hema Bashyam
bashyam@cshl.edu
516-367-6822
Cold Spring Harbor Laboratory
Source:Eurekalert

Related medicine news :

1. Live with Regis and Kelly Reveal A REVOLUTIONARY Pain Relief Method
2. Two Surgical Methods Equally Successful for Prostate Cancer
3. Psychiatrys main method to prevent mistaken diagnoses of depression doesnt work: NYU study
4. New testing method hints at garlics cancer-fighting potential
5. MSU scientists develop more effective method of predicting lead-poisoning risk
6. Blood Draw Dilemma: New Summit Health Article Helps Employers Choose the Best Screening Method
7. Worlds most useful tree provides low-cost water purification method for developing world
8. FDA, FSIS, CDC Collaborate on Methods to Measure Success of Food Safety Programs
9. Los Angeles Hair Transplant Cosmetic Surgeon Corrects Bad Hair Transplants With New Hair Restoration Method
10. An improved method for calculating tumor growth
11. New method to grow arteries could lead to biological bypass for heart disease
Post Your Comments:
*Name:
*Comment:
*Email:
(Date:4/28/2017)... NY (PRWEB) , ... April 28, 2017 , ... It's ... there are a number of illnesses that are unclear as to whether or not ... their heads. Bronchitis is one of these illnesses. So, FindaTopDoc took a look into ...
(Date:4/28/2017)... ... ... affects much more than energy – it also has mental and physical benefits. According to ... time, which can increase the risk of having a car accident. , This week ... to help you sleep better and feel better:, , Turn off ...
(Date:4/28/2017)... ... , ... The Radiology Business Management Association (RBMA) is pleased to ... election process has been in place since the RBMA was founded in 1968 with ... succeeds Jim Hamilton, MHA, CMM, FRBMA, as president. Dr. Dickerson the chief executive officer ...
(Date:4/28/2017)... , ... April 28, 2017 , ... ... America (UCAOA) and College of Urgent Care Medicine will host industry leaders for ... and speakers will help those in the industry adapt to the issues currently ...
(Date:4/28/2017)... ... April 28, 2017 , ... Yisrayl Hawkins, Pastor and Overseer at The House of Yahweh ... Creator responds to and which He does not. Yisrayl says with so many titles ... the true name, but he says with a little Scripture, backed with a lot of ...
Breaking Medicine News(10 mins):
(Date:4/19/2017)... EAST HANOVER, N.J. , April 19, 2017 ... study conducted by the National Heart, Lung, and ... Health (NIH) demonstrating that 58% of patients with ... at six months when treated with eltrombopag at ... treatment 1 . The study evaluated three sequential ...
(Date:4/18/2017)... April 18, 2017  Cardinal Health (NYSE: ... fiscal 2017 earnings per share (EPS) guidance and providing ... is in conjunction with this morning,s announcement of the ... and Nutritional Insufficiency businesses. Cardinal Health now ... will be at the bottom of its previous guidance ...
(Date:4/18/2017)... Viverae ® , a leader in workplace ... IBM ® Watson Campaign Automation, implementing behavioral messaging ... a personalized experience. Through digital engagement, the platform prompts ... real time. The enhanced experience drives engagement by focusing ... they are in their journey to health. ...
Breaking Medicine Technology: