Frequently Asked Questions
RNAi Introduction
shRNA libraries
- Who can receive shRNA clones from the RNAi Facility?
- How do I identify shRNA clones targeting my gene of interest?
- How do I request shRNA clones?
- What costs will be associated with requesting and receiving shRNA clones?
- My gene of interest is targeted by multiple shRNA clones, which clones should I assay?
- In what format will shRNA clones be supplied?
- How do I prepare shRNA plasmid DNA and glycerol stocks?
- Will the RNAi Facility have RNAi reagents available for species other than human?
- How was the Hannon shRNA library constructed?
Retrovirus
- How do I generate retrovirus from pSM2c vector DNAs?
- What safety measures must be in place to work with retroviral DNAs and retroviruses at Duke?
Troubleshooting
- What is plasmid recombination and why is it a concern?
- How can I minimize clone recombination?
- How do I know if a clone has recombined?
- How can I rescue a clone that exhibits recombination?
- I transformed a pSM2 clone into a standard E. coli strain but nothing grew - why?
- Where can I find the pSM2c vector sequence and map?
Other Services
RNAi Introduction
RNA interference (RNAi) is a natural regulatory mechanism conserved among many organisms in which double-stranded RNA (dsRNA) directs the post-transcriptional silencing of target genes in a sequence-specific manner. RNAi may also be employed as a powerful genetic analysis tool for the efficient silencing of target genes through the delivery of dsRNAs.
How does RNAi silence gene function?
The RNAi pathway is initiated by the processing of long dsRNAs of endogenous or exogenous origin by an endonuclease called Dicer. The resulting short interfering RNAs (siRNAs) are about 21 to 23 nucleotides in length and are incorporated into a nuclease complex, the RNA-induced silencing complex (RISC), which then targets and destroys message RNAs complementary to the siRNAs. Delivery of synthetic siRNAs or plasmid DNAs encoding short hairpin RNAs (shRNAs) into mammalian cells has been shown to elicit specific and robust RNAi-mediated gene silencing, or 'knock-down'.
shRNA or siRNA - which is right for my studies?
The 'functional' component of RNAi is the short interfering RNA, or siRNA. siRNAs can be synthesized, or can be the product of Dicer-processing of long dsRNAs or shRNAs. The choice between synthetic siRNAs and expressed shRNAs depends upon the requirements of the study. Synthetic siRNAs are delivered by transient transfection, and have been used successfully for one-off validation studies and highly-parallel cell-based screens. shRNAs differ in that they can be expressed from DNA vectors delivered by transient transfection or viral transduction. This allows for short- or long-term expression in a wide variety of cells, including difficult to transfect cells and in vivo models. DNA-encoded shRNAs can also be used for pooled shRNA selection studies. Please contact the RNAi Core for assistance with design of your studies.
shRNA Libraries
Who can receive shRNA clones from the RNAi Facility?
Along with the Expression Arrest™ human shRNA library, the RNAi core has purchased a license to distribute these clones to Duke investigators, and must maintain a log of all recipients of shRNA clones. Clones may not be distributed or shared off campus.
How do I identify shRNA clones targeting my gene of interest?
Search the Open Biosystems clone query or the RNAi Codex site for sequence verified hairpin constructs available in the pSM2c retroviral vector. More detailed information is available on the Request shRNA Clones page and in the Clone Request Instructions (PDF).
How do I request shRNA clones?
After identifying the desired clones (above), enter clone ID into the online shRNA Clone Request System. Detailed instructions are available here (Clone Request Instructions PDF).
What costs are associated with requesting and receiving shRNA clones?
Individual or sets of clones will be made available to Duke researchers for $30 per clone to cover expenses associated with maintaining and distributing the libraries. Additionally, purified plasmid DNA from pooled human shRNA clones is available for $5 per 1ug of DNA (scalable). Pooled DNAs are available in 33 sets of ~2000 clones, or further combined into more complex pools.
My gene of interest is targeted by multiple shRNA clones, which clones should I assay?
The Hannon-Elledge shRNA library has, on average, greater than 2 shRNA clones targeting each gene. This redundancy is due to the fact that RNAi-mediated knock-down is variable, and some clones may be less effective than others. Therefore, it is highly recommended that you obtain and assay all available shRNA clones targeting your gene or genes of interest. This relatively minor investment in multiple clones and parallel validation will greatly increase your chances for success.
In what format will shRNA clones be supplied?
The shRNA libraries are stored as bacterial glycerol stocks of individual shRNA plasmid DNAs. Upon receiving a clone request, frozen bacterial stock will be scraped into culture media and prepared for distribution. Large requests may be distributed in 96-well format. We recommend processing clones immediately upon receipt (grow and prep DNA or store as frozen glycerol stock) to minimize loss of viability. You will be charged for repeat clone requests.
How do I prepare shRNA plasmid DNA and glycerol stocks?
Protocols are available for preparation and characterization of shRNA plasmid DNA (PDF file) in individual clone and 96-well formats, as well as best practices for maintenance and storage of clones. For additional protocols, visit the RNAi Protocols page.
Will the RNAi Facility have RNAi reagents available for species other than human?
The RNAi Facility currently maintains and distributes only the Hannon/Elledge human retroviral shRNA library. However, we will continue to assess needs and investigate the possibility of adding other RNAi collections, including those targeting other species (for example, mouse) or in other delivery systems (for example, lentivirus).
Additionally, we have negotiated a Duke-wide 'Custom siRNA/shRNA Vector Construction Service' agreement through an external vendor to provide shRNA synthesis, cloning into expression vector, and sequence verification (see Question 17 below).
How was the Hannon shRNA library constructed?
In early 2004, Paddison et al described their strategy and vector components used to prepare their shRNA libraries in the pSHAG-MAGIC1 vector (Short Hairpin Activated Gene silencing - Mating Assisted Genetically Integrated Cloning System, version 1). Further understanding of RNAi mechanisms and how to exploit RNAi pathways for enforcing silencing in mammals led to version 2 of the library in pSHAG-MAGIC2c (pSM2c; Silva et al.). This second generation of shRNAs is modeled after endogenous microRNAs and incorporates two primary improvements:
- The vector was modified to mimic the natural miR-30 microRNA transcript, resulting in up to 12-fold increase production of mature synthetic microRNAs.
- The shRNAs were designed using improved bioinformatics derived from empirical data to target sequences that maximize efficiency by directing preferential incorporation of the correct strand into RISC (Cleary et al.).
Tests of these strategies on components of the proteasome revealed a large increase in both the percentage of clones that functioned and the strength of the phenotype that they created. The results of careful titration experiments of plasmid and RNA transfections followed by quantification of small RNAs in the cell suggest that an equivalent of at least 40 nM siRNA is achieved. This is well within the effective range of a good siRNA.
For details and protocols for the generation of the Hannon-Elledge shRNA libraries, visit RNAi Central.
Retrovirus
How do I generate retrovirus from pSM2c vector DNAs?
Instructions for packaging of retroviral particles (from Open Biosystems) are available on the Protocols page.
Alternatively, the Phoenix retroviral packaging line can be used.
What safety measures must be in place to work with retroviral DNAs and retroviruses at Duke?
From the Duke Occupational & Environmental Safety Office (OESO)
"Experiments involving the utilization of recombinant DNA may require approval by the Duke University Institutional Biosafety Committee (IBC) prior to submission to outside agencies and the initiation of experimentation. PIs should obtain proper Recombinant DNA forms.
All research involving viral vector expression systems (including retrovirus) must be registered by completing and submitting the Duke Viral Vector Registration form. An IBC representative will confirm that the appropriate handling precautions have been prescribed after reviewing the information included in the form.
The RNAi Facility is required to reports the distribution of retroviral vector DNAs to the Duke Biosafety Office, so please ensure the proper SOPs and approvals are in place for your studies.
Troubleshooting
What is plasmid recombination and why is it a concern?
It is well documented that retroviral plasmid DNAs are inherently prone to recombination through the long terminal repeat (LTR) regions. Recombination appears to be exacerbated by MAGIC-ready bacterial strains containing F' episome. Thus, you will receive clones in a non-MAGIC bacterial strain, DH10βpir116.
The nature of the recombination in the pSM2c retroviral vector is thought to be an LTR to LTR recombination, resulting in a complete loss of the shRNA sequence as well as the chloramphenicol and the puromycin resistance genes. The recombinant product may contain oriT, the RK6γ origin, the kanamycin resistance gene, and a portion of the LTRs. Unfortunately, it appears that despite the loss of chloramphenicol resistance, you cannot use chloramphenicol to select against the recombinant since it resides in cells with a complete (non-recombined) plasmid. It is therefore critical to stringently adhere to recommended growth conditions when culturing viral vectors in E. coli in order to reduce recombination. Fortunately, the pSM2 retroviral vector produces little recombinant background product under careful growth and handling conditions.
How can I minimize clone recombination?
To ensure clone integrity, it is recommended to streak this starter culture onto LB/agar containing fresh Cloramphenicol and to characterize multiple isolated colonies. Standard precautions include growth in low-recombination (and replication competent) bacterial strains such as PirPlus (DH10βpir116) E. coli, minimizing the duration of bacterial growth steps, adding freshly prepared antibiotics (especially chloramphenicol), and characterizing multiple clone isolates. Detailed instructions are provided on the Protocols page.
How do I know if a clone has recombined?
The quickest way to identify plasmid recombination is by doing a plasmid prep and looking at the product on a gel. EcoRI, XhoI, and XbaI linearize the intact vector but do not cut the recombination product, and HindIII cuts the recombinant once and the complete plasmid twice (see protocols). There appear to be two predominant mechanisms of recombination with the pSM2c vector, resulting either in smaller (1.7 and 1.2kb) or larger (~12-14kb) than expected plasmid DNA (7.1kb).
12-14kb recombination product:
This 'uber' plasmid is thought to be the result of a plasmid fusion event, producing a larger than expected shRNA plasmid. While this may not be ideal, it has been demonstrated by DNA sequencing and in transient knock-down studies that this uber plasmid encodes the shRNA sequence and is capable of eliciting potent knock-down. It has been suggested that this vector may also be appropriately packaged into retrovirus.
1.7/1.2kb recombination product:
The primary LTR to LTR recombination of pSM2 is thought to cause a complete loss of the shRNA sequence as well as the chloramphenicol and the puromycin resistance genes, resulting in a visible 1.7kb (and somtimes 1.2kb) product on a gel. This recombination product is typically a minor contaminant in a prep of intact plasmid, and low levels of contamination may be tolerable. The following is a gel image of uncut and EcoRI-cut pSM2-shRNA DNA with recombination products visible at ~1.7 kband ~1.2kb.
How can I rescue a clone that exhibits 1.7/1.2kb recombination?
Depending upon the intended use of an shRNA vactor, some level of recombination may be tolerable. For example, 1/7/1.2kb recombination products of pSM2 should lack Puro-resistance, and thus will not produce productive and Puro-resistant retrovirus. Additionally, low levels of contamination with recombination products in a predominantly intact plasmid prep may be acceptable (though not ideal) for transient transfection studies.
To 'clean up' a sample of recombinant bands you have two options. First, you can gel isolate the pSM2 non-recombined DNA from the recombinant band and re-transform into E. coli. Alternatively, you can take the recombined sample, without gel isolation (you must dilute DNA to ensure one plasmid per cell), and then re-transform into E. coli and select with Chloramphenicol. If you retransform into the MAGIC strain with this particular vector it will recombine again in the future, if you use the more stable strain (DH10βpir116) which is not MAGIC compatible, it appears to be stable through multiple rounds of growth.
I transformed a pSM2 clone into a standard E. coli strain but nothing grew - why?
Plasmid pSM2c contains the RK6γ conditional origin of replication, and requires pir1 expression by host strain for propagation. Thus, a pir1 strain such as PirPlus (DH10βpir116) E. coli must be used for propagation.
Where can I find the pSM2c vector sequence and map?
pSM2c vector sequence data and vector map is available here.
Other Services
Can the RNAi Facility help with construction of custom shRNA clones?
The RNAi Facility has negotiated a Duke-wide 'Custom siRNA/shRNA Vector Construction Service' agreement through an external vendor. This agreement provides preferred pricing and rapid turn-around on shRNA insert synthesis (you design insert sequence), construction (you provide vector and map), sequence verification of insert, and vector DNA. For more information, contact Tom Burke.