The RNAi Consortium
Encyclopedia
The RNAi Consortium, or TRC, is a public-private partnership
whose mission is to create libraries of small hairpin RNA
s (shRNAs) for 15'000 human and 15'000 mouse gene
s. These libraries should help the scientific community to analyse gene function by RNAi
. The consortium is based at the Broad Institute
of the MIT and Harvard University
, and includes 6 MIT- and Harvard-associated institutions and 5 international life sciences organizations. Verified RNAi clones and entire libraries are made available both by Sigma-Aldrich
and Open Biosystems.
transcript
from each NCBI
gene. They should be 21mers, be at least 25bp from start of the coding sequence and no closer than 150bp from its end. Candidates are scored based on various empirical
rules (see the Broad Institute's web site for a complete list http://www.broad.mit.edu/genome_bio/trc/rules.html) and then BLAST
ed against 2 transcriptome
sets. Hairpins that are unique for a Unigene
cluster and a RefSeq NM identifier are preferred. Lastly, the candidates are spaced to have 1 hairpin in the 3' untranslated region and 4 in the coding sequence.
pLKO1, which is a multipurpose plasmid
that can be propagated in bacteria
, transfect
ed into mammal
ian cell lines or used for generation of lentivirus
es. It contains resistance gene
s against ampicillin
and puromycin
.
Public-private partnership
Public–private partnership describes a government service or private business venture which is funded and operated through a partnership of government and one or more private sector companies...
whose mission is to create libraries of small hairpin RNA
Small hairpin RNA
A small hairpin RNA or short hairpin RNA is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA uses a vector introduced into cells and utilizes the U6 or H1 promoter to ensure that the shRNA is always expressed...
s (shRNAs) for 15'000 human and 15'000 mouse gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
s. These libraries should help the scientific community to analyse gene function by RNAi
RNAI
RNAI is a non-coding RNA that is an antisense repressor of the replication of some E. coli plasmids, including ColE1. Plasmid replication is usually initiated by RNAII, which acts as a primer by binding to its template DNA. The complementary RNAI binds RNAII prohibiting it from its initiation role...
. The consortium is based at the Broad Institute
Broad Institute
The Broad Institute is a genomic medicine research center located in Cambridge, Massachusetts, United States. Although it is independently governed and supported as a 501 nonprofit research organization, the institute is formally affiliated with the Massachusetts Institute of Technology, Harvard...
of the MIT and Harvard University
Harvard University
Harvard University is a private Ivy League university located in Cambridge, Massachusetts, United States, established in 1636 by the Massachusetts legislature. Harvard is the oldest institution of higher learning in the United States and the first corporation chartered in the country...
, and includes 6 MIT- and Harvard-associated institutions and 5 international life sciences organizations. Verified RNAi clones and entire libraries are made available both by Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich Corporation , is a life science and high technology company with over 7,600 employees and operations in 40 countries. Its chemical and biochemical products and kits are used in scientific research, biotechnology, pharmaceutical development, the diagnosis of disease, and as key...
and Open Biosystems.
Hairpin selection
A set of candidate hairpins are selected based on the 1st RefseqRefSeq
The Reference Sequence database is an open access, annotated and curated collection of publicly available nucleotide sequences and their protein products. This database is built by National Center for Biotechnology Information , and, unlike GenBank, provides only single record for each natural...
transcript
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
from each NCBI
National Center for Biotechnology Information
The National Center for Biotechnology Information is part of the United States National Library of Medicine , a branch of the National Institutes of Health. The NCBI is located in Bethesda, Maryland and was founded in 1988 through legislation sponsored by Senator Claude Pepper...
gene. They should be 21mers, be at least 25bp from start of the coding sequence and no closer than 150bp from its end. Candidates are scored based on various empirical
Empirical method
The empirical method is generally taken to mean the approach of using a collection of data to base a theory or derive a conclusion in science...
rules (see the Broad Institute's web site for a complete list http://www.broad.mit.edu/genome_bio/trc/rules.html) and then BLAST
BLAST
In bioinformatics, Basic Local Alignment Search Tool, or BLAST, is an algorithm for comparing primary biological sequence information, such as the amino-acid sequences of different proteins or the nucleotides of DNA sequences...
ed against 2 transcriptome
Transcriptome
The transcriptome is the set of all RNA molecules, including mRNA, rRNA, tRNA, and other non-coding RNA produced in one or a population of cells.-Scope:...
sets. Hairpins that are unique for a Unigene
Unigene
Unigene Laboratories is a biopharmaceutical company, engaged in the research and development of peptides for medical purposes. The company was founded in 1980 and is located in New Jersey....
cluster and a RefSeq NM identifier are preferred. Lastly, the candidates are spaced to have 1 hairpin in the 3' untranslated region and 4 in the coding sequence.
Hairpin vector
Selected hairpins are cloned into the vectorViral vector
Viral vectors are a tool commonly used by molecular biologists to deliver genetic material into cells. This process can be performed inside a living organism or in cell culture . Viruses have evolved specialized molecular mechanisms to efficiently transport their genomes inside the cells they infect...
pLKO1, which is a multipurpose plasmid
Plasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
that can be propagated in bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
, transfect
Transfection
Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
ed into mammal
Mammal
Mammals are members of a class of air-breathing vertebrate animals characterised by the possession of endothermy, hair, three middle ear bones, and mammary glands functional in mothers with young...
ian cell lines or used for generation of lentivirus
Lentivirus
Lentivirus is a genus of slow viruses of the Retroviridae family, characterized by a long incubation period...
es. It contains resistance gene
Antibiotic resistance
Antibiotic resistance is a type of drug resistance where a microorganism is able to survive exposure to an antibiotic. While a spontaneous or induced genetic mutation in bacteria may confer resistance to antimicrobial drugs, genes that confer resistance can be transferred between bacteria in a...
s against ampicillin
Ampicillin
Ampicillin is a beta-lactam antibiotic that has been used extensively to treat bacterial infections since 1961. Until the introduction of ampicillin by the British company Beecham, penicillin therapies had only been effective against Gram-positive organisms such as staphylococci and streptococci...
and puromycin
Puromycin
Puromycin is an antibiotic that is a protein synthesis inhibitor by inhibiting translation.-Inhibition of translation:Puromycin is an aminonucleoside antibiotic, derived from the Streptomyces alboniger bacterium, that causes premature chain termination during translation taking place in the...
.