This is the simplest format of RNAi. One of the biggest hurdles for achieving effective RNAi with siRNA is that many cells are difficult to transfect. An RNAi experiment is typically considered successful when the target gene expression is reduced by >70%, a threshold not attained by many types of cells due to their low transfection efficiency. Another drawback of using synthetic siRNA is the limited duration of post-transfection effects, with gene silencing activities peaking at around 24 hours, and then diminishing within 48 hours.
To improve specific activity, reduce cell toxicity, and off-target effects, chemically synthesized siRNAs should be HPLC purified. Allele Biotech offers commercial siRNA with the following features:
shRNA can be introduced via DNA plasmid, linear template, or packaged retroviral/lentiviral vectors. Using any form of DNA construct, except the PCR template format found in Allele’s LineSilence platform, requires creating DNA constructs and sequence verification; a taxing work load if multiple genes need to be studied. However, once the constructs are made, they can be reproduced easily and inexpensively. It is difficult to directly compare the effectiveness of siRNA versus shRNA on a per molecule basis because RNA polymerase III (Pol III) promoters such as U6 or H1 commonly used to express shRNAs can make thousands of copies of shRNA from a single DNA template. However when both siRNA and shRNA are produced the same way, e.g. synthesized chemically, shRNA is reported to be somewhat more effective.
Allele Biotech has been granted multiple patents (US patents 7,294,504, 7,422,896, 7,625,750, and China patent CN02828345.7) on DNA-based, RNA polymerase III driven shRNA platforms (e.g. H1, U6) that are popularly used as gene silencing tools. Compared to other shRNA-expressing plasmids, Allele’s SilenCircle platform design allows high-level expression of shRNA. RNAi transcripts expressed by this method result in sequence identifications that are precisely as desired (i.e. without extra bases introduced by cloning sites), thereby reducing non-specific effects. Additional features include:
LineSilenceTM RNAi cassettes (invented in 2001, under US patents 7,294,504, 7,422,896, 7,625,750, and China patent CN02828345.7) use engineered human U6 polymerase III promoter and modified terminator for high-level, precise siRNA expression inside mammalian cells. Cloning is not necessary. It takes only a few hours to generate RNAi reagents ready for transfection. It is particularly suitable for screening for effective RNAi targets with convenience at a low cost. If desired, these cassettes can be easily cloned into any vector, e.g. plasmid or virus, for amplification or other special purposes. Additional features include:
Viruses that can integrate into the chromosomes of most host cells are often used to carry shRNA or miRNA for RNAi screening because of their high transduction rates. Most researchers use standard cloning procedures when trying to insert shRNA templates into lentiviral vectors, i.e. anneal a pair of long oligos with sticky ends and ligate the dsDNA into a linearized plasmid with compatible overhangs. However, since typical lentiviral vector plasmids have terminal repeats and are relatively large, when ligated to hairpin sequence-containing shRNA templates, recombination often occurs inside the bacteria resulting in smaller plasmids. Allele Biotech has developed a highly efficient ligation and sequencing-free process for shRNA library production as part of an NCI project in 2010. Features include:
Even though there have been several commercial sources that provide designed or validated RNAi viral plasmids or viral particles, most of them suffer low interference efficiency or low transduction efficiency of virus. That has been a reason why researchers resort to validate RNAi target sequences by themselves or through custom services. With Allele Biotech's powerful and unique viral packaging services, patent-protected RNAi platform, and state-of-the-art fluorescent protein markers, we provide a full service for RNA interference, including RNAi expression viral packaging, fluorescent protein-based RNAi validation, and even large scale RNAi screening.