Achieve long-term, inducible, and in vivo gene silencing while minimizing off-target effects with shRNA reagents by Dharmacon™

microRNA-adapted shRNAs

Simple hairpin shRNAs

  • TRC lentiviral shRNA

    Lentiviral shRNA collection from The RNAi Consortium (TRC) for human and mouse cells

Short hairpin RNA (shRNA) sequences are encoded in a DNA vector that can be introduced into cells via plasmid transfection or viral transduction. Because the shRNA expression cassettes can be incorporated into viral vector systems, including lentivirus, they can integrate into the host genome for the creation of stable cell lines. Additionally, when used in combination with one of several viral delivery systems, they can be delivered into difficult-to-transfect primary cells and used for in vivo applications. Based on the delivery method and vector design, vector-based shRNAs can allow for long-term (or inducible) down-regulation of target genes.

The performance of shRNA is influenced by many factors including the efficiency of transduction or transfection, the promoter driving expression of the shRNA and epigenetic modifications (which can lead to silencing of shRNA expression). Further, the influence that each of these factors have on vector performance can differ depending on the cell line or cell type. When planning an experiment using shRNA the available vector options, including; the shRNA design to be used, the vector features (e.g.,promoter), and the method of delivery should all be taken into consideration based on the requirements of the experiment.

shRNA design

shRNA design is typically divided into two formats, the simple stem-loop shRNA and the microRNA-adapted shRNA.

Simple stem-loop shRNA

Basic shRNAs are modeled on precursor microRNA (pre-miRNA), and are cloned into viral vectors where they are transcribed under the control of RNA Polymerase III (Pol III) promoters. shRNAs are produced as single-strand molecules of 50–70 nucleotides in length, and form stem loop structures consisting of a 19-29 base-pair region of double-strand RNA (the stem) bridged by a region of single-strand RNA (the loop) and a short 3’ overhang. Once transcribed, shRNAs exit the nucleus, are cleaved at the loop by the nuclease Dicer in the cytoplasm, and enter the RISC to direct cleavage and subsequent degradation of complementary mRNA.

microRNA adapted shRNA

A microRNA-adapted shRNA consists of a shRNA stem structure with microRNA-like mismatches surrounded by the loop and flanking sequence of an endogenous microRNA. microRNA-adapted shRNAs are transcribed from RNA Polymerase ll (Pol ll) promoters, cleaved by the endogenous RNase III Drosha enzyme in the nucleus, and then exported to the cytoplasm where they are processed by Dicer and loaded into the RISC complex. Studies have suggested that the use of a microRNA scaffold, which is processed by both Drosha and Dicer, may promote more efficient processing and reduce toxicity for in vivo RNAi.

RNA interference and manipulation

rna  interference and manipulation

Figure 1. shRNA approaches include the introduction of genetically engineered viral vectors or plasmid-based vectors expressing silencing sequences embedded in an endogenous microRNA scaffold (1) or simple stem-loop shRNA (2). Expressed sequences (1 and 2, shown in blue) enter the endogenous pathway at an early stage and are efficiently processed into potent silencing molecules using the endogenous microRNA mechanism. All of these approaches lead to target mRNA cleavage (shown in purple) and gene silencing.

References and recommended reading

  1. Liu, Z. et al. (1997) A systematic comparison of relative promoter/enhancer activities in mammalian cell lines.Analytical Biochem. 246(1):150-152.
  2. Ramezani, A. et al. (2000) Lentiviral vectors for enhanced gene expression in human hematopoietic cells.Molecular Therapy 2(5):458-469.
  3. Brummelkamp, T.R. et al. (2002) A system for stable expression of short interfering RNAs in mammalian cells.Science 296(5567):550-553.
  4. Paddison, P.J. et al. (2002) Stable suppression of gene expression by RNAi in mammalian cells. PNAS 99(3):1443-1448.
  5. Paul, C.P. et al. (2002) Effective expression of small interfering RNA in human cells. Nature Biotechnology 20(5):505-508.
  6. Bartel, D.P. (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116(2):281-297.
  7. Gregory, R.I. et al. (2005) Human RISC couples microRNA biogenesis and post-transcriptional gene silencing.Cell 123(4):631-640.
  8. Kim, V.N. (2005) MicroRNA biogenesis: coordinated cropping and dicing. Nature Reviews, Molecular Cell Biology 6(5):376-385.
  9. Silva, J.M. et al. (2005) Second-generation shRNA libraries covering the mouse and human genomes. Nature Genetics 37(11):1281-1288.
  10. Siolas, D. et al. (2005) Synthetic shRNAs as potent RNAi triggers. Nature Biotechnology 23(2):227-231.
  11. Grimm, D. et al. (2006) Fatality in mice due to oversaturation of cellular microRNA/ short hairpin RNA pathways. Nature 441:537-541.
  12. Hong, S. et al. (2007) Functional analysis of various promoters in lentiviral vectors at different stages of in vitro differentiation of mouse embryonic stem cells. Molecular Therapy 15(9):1630-1639.
  13. Luo, B. et al. (2008) Highly parallel identification of essential genes in cancer cells. PNAS 105(51):20380-20385.
  14. McBride, J.L. et al. (2008) Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: implications for the therapeutic development of RNAi. PNAS 105(15):5868-5873.
  15. Schlabach, M.R. et al. (2008) Cancer proliferation gene discovery through functional genomics. Science 319(5863):620-624.
  16. Silva, J.M. et al. (2008) Profiling essential genes in human mammary cells by multiplex RNAi screening. Science 319(5863): 617-620.
  17. Zender, L. et al. (2008) An oncogenomics-based in vivo RNAi screen identifies tumor suppressors in liver cancer. Cell 135(5):852-864.
  18. Gumireddy, K. et al. (2009) KLF17 is a negative regulator of epithelial-mesenchymal transition and metastasis in breast cancer. Nature Cell Biology 11(11):1297-1304.
  19. Maier, B. et al. (2009) A large-scale functional RNAi screen reveals a role for CK2 in the mammalian circadian clock. Genes and Development 23:708-718.
  20. Mullenders, J. et al. (2009) Candidate biomarkers of response to an experimental cancer drug identified through a large-scale RNA interference genetic screen. Clinical Cancer Research 15(18):5811-5819.
  21. Peng, J. et al. (2009) Jarid2/Jumonji coordinates control of PRC2 enzymatic activity and target gene occupancy in pluripotent cells. Cell 139(7):1290-1302.
  22. Yeung, M.L. et al. (2009) A genome-wide short hairpin RNA screening of jurkat T-cells for human proteins contributing to productive HIV-1 replication. Journal of Biological Chemistry 284(29):19463-19473.
  23. Beer, S. et al. (2010) Low-level shRNA cytotoxicity can contribute to MYC induced hepatocellular carcinoma in adult mice. Molecular Therapy 18(1):161-170.
  24. Du, W. et al. (2010) Cytoplasmic FANCA-FANCC complex interacts and stabilizes the cytoplasm-dislocalized leukemic Nucleophosmin Protein (NPMc). Journal of Biological Chemistry 285(48):37436-37444.
  25. Rato, S. et al. (2010) Novel HIV-1 knockdown targets identified by an enriched kinases/phosphatases shRNA library using a long-term iterative screen in Jurkat T-cells. PLoS One 5(2):e9276.
  26. Smolen, G.A. et al. (2010) A genome-wide RNAi screen identifies multiple RSK-dependent regulators of cell migration. Genes and Development 24(23):2654- 2665.
  27. Fellmann, C. et al. (2011) Functional identification of optimized RNAi triggers using a massively parallel sensor assay. Molecular Cell 41(6):733-746.
  28. Montgomery, R.L. et al. (2011) Therapeutic inhibition of miR-208a improves cardiac function and survival during heart failure. Circulation 124(14):1537-1547.
  29. Pan, Q. et al. (2011) Disturbance of the microRNA pathway by commonly used lentiviral shRNA libraries limits the application for screening host factors involved in hepatitis C virus infection. FEBS Letters 585:1025-1030.
  30. Ying, M. et al. (2011) Kruppel-like family of transcription factor 9, a differentiation-associated transcription factor, suppresses Notch1 signaling and inhibits glioblastoma-initiating stem cells. Stem Cells 29(1):20-31.

Which shRNA reagent is best for you?

Use the table below to assist you in determining the right shRNA reagents for your experimental needs.

Species Human, Mouse, Rat Human, Mouse Human Human, Mouse
Promoter Choice of 7 constitutive* and 4 inducible promoters Human CMV Pol II TRE-min-CMV U6 Pol III
Vector backbone Lentiviral Lentiviral Lentiviral Lentiviral
Stem-loop format MicroRNA-adapted MicroRNA-adapted MicroRNA-adapted Simple
Fluorescent reporter gene GFP/RFP GFP RFP
Guaranteed silencing**
In vivo RNAi
Create stable cell lines
Recommended for primary and non-dividing cells
Inducible expression
Whole genome library availability Human, Mouse, Rat Human, Mouse Human Human, Mouse
Formats Bacterial glycerol stock High-titer lentiviral particles Gene families and pathways Bacterial glycerol stock Arrayed library format Gene families and pathways High-titer lentiviral particles shRNA Starter Kit Bacterial glycerol stock Arrayed library format Gene families and pathways shRNA Starter Kit Bacterial glycerol stock Arrayed library format Gene families and pathways

*Some promoter options may only be available as custom products or upon request.

**For SMARTvector, GIPZ, and TRIPZ lentiviral shRNAs, at least one out of three constructs is guaranteed to reduce target mRNA levels by 70% or more when used in combination with the appropriately matched non-targeting and positive controls.

shRNA controls

Critical to any gene silencing experiment, shRNA controls enable accurate interpretation for reliable, reproducible results

Pooled shRNA libraries

A pooled lentiviral screen can be performed to identify genes that regulate cellular responses and signaling pathways, or to discover novel gene functions. Pooled screening libraries can consist of as few as 50 constructs up to many thousands. In contrast to the costly automated techniques that are required to screen using individually arrayed reagents, pooled screening libraries allow the researcher to transduce and screen a population of cells within a few tissue culture dishes.

  • SMARTvector lentiviral shRNA pooled libraries

    High-titer pooled screening libraries of constitutive SMARTvector Lentiviral shRNAs for pre-defined gene libraries in human, mouse, and rat. Choose from seven promoters and three reporter options to select the best format for your experiment.

  • SMARTvector inducible lentiviral shRNA pooled libraries

    High-titer pooled screening libraries of SMARTvector Inducible Lentiviral shRNAs for pre-defined gene libraries in human, mouse, and rat. Choose from four promoters and two reporter options to select the best format for your experiment. 

  • Custom pooled lentiviral screening libraries

    Select from a range of our algorithm-optimized reagents to develop the ideal pooled lentiviral screening resource for CRISPR-Cas9 knockout or RNAi knockdown screens.

Arrayed shRNA libraries

Delivered as glycerol stocks in 96-well format; these libraries are ideal for arrayed screening formats to elucidate gene activity and study specific pathways or gene families.