About three months after the Integrative Cancer Biology Program at the Massachusetts Institute of Technology first activated its online database of experimentally validated siRNA and shRNA sequences, plans are underway to expand the number of labs contributing to the effort.
The so-called MIT/ICBP siRNA database, which MIT’s ICBP released in July, is currently limited to sequences provided by the lab of Phillip Sharp, according to Mary Lindstrom, who manages the Sharp lab and oversees the database.
Over the past month, however, Lindstrom has been identifying researchers from other MIT labs within the ICBP who have published papers in which “they used siRNA and shRNA reagents … and had good data showing mRNA or protein knockdown,” she told BioInform’s sister publication RNAi News this week.
“The stage I’m at now is starting to approach each of those researchers to see, first, if they’d be interested in submitting those sequences that are published [to the database], and then going on to [discuss submitting] sequences that are still in the works,” she said.
Lindstrom added that plans are underway to possibly begin accepting sequences from labs at other ICBP-affiliated institutions. There are nine research institutes under the ICBP program, which is funded by the National Cancer Institute. These include MIT, Case Western Reserve University, Dana Farber Cancer Institute, Duke University, Lawrence Berkeley National Laboratory, Massachusetts General Hospital, Ohio State University, Stanford School of Medicine, and Vanderbilt University Medical Center.
“There is an ICBP steering committee meeting next month where I’ll be presenting the database in a poster form to determine if there is interest outside of MIT for this resource and contributing to it,” she said.
However, it wasn’t immediately clear whether the ICBP at MIT will go one step further and begin taking sequence submissions from labs outside of the ICBP.
“We haven’t fully decided if we’re going to end up expanding this to non-ICBP researchers,” Lindstrom noted, adding that “we are very interested to see if there is interest by outside labs.
“If there does seem to be sufficient interest by [outside] people to submit sequences, then [we would begin determining] how we would accept those submissions,” she said.
Currently, for sequences to be included into the database the ICBP at MIT requires information on the type of knockdown experiment conducted, the cell lines in which the RNAi reagents were tested, and details on how mRNA or protein knockdown was determined, Lindstrom explained.
She said this “same type of quality control” would be applied to all non-ICBP labs wishing to submit sequences.
This would go for any sequences derived from industry sources, as well.
“We’re not limited to sequences that have been developed by researchers here,” Lindstrom said. “So if a person buys a sequence from a company, they test it and show in their cell lines that this works, then they can submit those sequences.”
Seeking Validation
The NCI formed the ICBP in 2004 to “gain new insights into the development and progression of cancer through a systems-wide approach [that involves applying] an integrative and multi-disciplinary effort among all fields of cancer research.” It also sought to incorporate “a spectrum of new technologies such as genomics, proteomics, and molecular imaging to generate computer and mathematical models that could predict the cancer process.”
Several labs within the MIT ICBP focus on RNAi research, and the siRNA database was developed to support this work. “With the increasing number of experimentally verified siRNAs and shRNAs created and used by members of the MIT community, it has become desirable to have a comprehensive, easily accessible database to store and distribute tested siRNA/shRNA information,” the ICBP states on the database website.
“When people develop siRNA or shRNA reagents, even with the most robust computational methods currently available, there is a pretty good chance that the reagents will not work [and] that you will not get substantial knockdown of mRNA or protein,” Lindstrom said.
“The only way to really tell if the reagent will work is to test it experimentally,” she added. “The idea behind our resource is that we will collect information on those siRNA or shRNA reagents that have actually been tested in the lab and shown to produce either mRNA or protein knockdown.
“So someone interested in knocking down a particular gene can come to our resources and be able to determine if someone here at MIT has been able to knock down that gene previously, and if so, using what reagent,” she said.
Right now, the database is limited to sequences for two collections of genes. The first is a set of “control genes” commonly used for gene knockdown experiments such as luciferase or EGFP, Lindstrom said.
The second comprises sequences for other genes that have been used to study the siRNA mechanism, including CXCR4, which “is the gene that we have used extensively to try to figure out how gene knockdown works in cells,” she added.
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“If there does seem to be sufficient interest by [outside] people to submit sequences, then [we would begin determining] how we would accept those submissions.” |
However, the number of sequences within the database is expected to grow significantly once other labs within MIT’s ICBP program and outside ICBP affiliates begin contributing.
“Some of the [sequences for] genes of interest that we’re attempting to get other labs … to submit include [ones for] some of the genes that are actually within the RNAi pathway such as Dicer and Drosha, as well as pretty much anything else the people in the ICBP labs are working on,” Lindstrom said.
Further, while the database is currently populated only by sequences against mammalian genes, Lindstrom said she would accept siRNA and shRNA sequences used in other organisms.
“We’re open to any type of sequence … because we figure that this resource could be used by people who are specifically interested in cancer research, as well as people who are interested more in the global RNAi pathway and understanding it, which would include some of the work in C. elegans,” for instance, she said.
Also contributing to the robustness of the siRNA/shRNA database is a portal to the National Center for Biotechnology Information’s Probe Database of functional genomics reagents.
“We’ve worked with the NCBI to streamline submissions from our database into their database, which gives us an extra level of information because the sequences submitted to them become integrated with all of their genomics information,” Lindstrom said.
A version of this article appears in the current issue of BioInform sister publication RNAi News.