Making primers

http://allelebiotech.com/blogs/2011/04/making-primers/

We often hear that oligos (primers) are a simple product, and they should work every single time. From a producer’s point of view, oligos are arguably the most complicated products among common molecular biology reagents, at least in terms of the number of chemical steps required. DNA synthesis starts from the 3’ end to the 5’ end (opposite to DNA polymerases) on a solid support (e.g. CPG beads). For the addition of each base, the process begins by removing a protection group via “Deblocking”; then activating the last base for coupling with an “Activator”, adding the current nucleotide in the chemical form of a phosphoramidite (4 times in our protocol), blocking un-reacted openings with “Capping A and B solutions” (again 4 times each), forming bonds between bases by oxidization with Iodine, then looping all the way back to the beginning of the cycle. Many of these chemicals are either highly sensitive to moisture or have a short shelf life (can go bad any time).

Coming back from a seminar, sitting at the lab desk, you know you have a new idea and some cloning to do, and of course, it must be done tomorrow. The first thing you do is to send in some oligo sequences online to a local synthesis company late in the afternoon after looking at some maps and sequences. Around noon the next day, the oligos will be delivered in person to your hands. Most times everything will just work out fine as far as experiments involving primers are concerned; others you get stuck here and there along the way of cloning. Chances are you have run into problems with primers not giving PCR signals or clones with mistakes in the primer regions at some points in your research career. Even if this has only happened a few times, the memory, as well as the dissatisfaction and anger, can last for quite some time.

Between ordering and delivering, oligos are made overnight; they are then post-synthesis processed (requiring several hours starting in the early morning), OD’ed, and concentration adjusted. Given that the machine completes the run without any problems (power or computer related), and none of the chemicals run out, the best quick indicator of a good run is a color change from the protection group removed by Deblock at the last base. If there is visible amount of blocking group at the end of the synthesis, as reflected by an orange color from a Trityl group, it is likely that the synthesis was efficient till the end. Unfortunately, the efficiency of adding each base is never 100%–accumulations of missing or, at a lower percentage, mistaken bases will add up, especially in long oligos. Purification will remove some of the oligos with deletions, but not all of the bad oligos. MASS analysis will help determine the approximate percentage of bad oligos, but it will require time and cost not typically chosen by customers. It is our hope that understanding how oligos are made will help with more effective use of oligos when you order oligos, conduct experiments using oligos, or clone with oligos.

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Dealing with Interferon Response When Doing RNAi

From AlleleBlog: http://allelebiotech.com/blogs/2010/09/dealing-with-interferon-response-when-doing-rnai/

Off-target effects are a major problem when using RNA interference (RNAi) to silence genes in mammalian systems. One potential source of off-target effects, by either transfected siRNA duplexes or transcriptionally expressed shRNAs, is the inadvertent activation of the interferon response. There are several steps that can be taken to deal with this problem.

Delivery
Interferon response is more likely when high levels of siRNA are used; it is important to transfect the minimum amount of the siRNA duplex that gives rise to a specific RNAi response, as assessed by the level of expression of the target mRNA and/or protein. The level of stable shRNA expression achieved by using lentiviral or retroviral vectors is comparatively modest. Unless very high levels of shRNA expression are achieved, for example, by using highly transfectable cells and a very efficient shRNA expression plasmid, nonspecific activation of the innate immune response are less likely to be induced.

Design
Previous work has shown that the interferon response is induced by dsRNAs of ?30 bp in length and that perfect dsRNAs of as little as 11 bp in length can produce a weak induction. One possible approach to solving the problem of nonspecific activation of the cellular interferon response is to design the siRNA duplex or shRNA precursor so that it does not contain any stretches of perfect dsRNA of ?11 bp.

Detection
If activation of the interferon response remains a concern, it is possible to routinely check for this effect during the course of an RNAi experiment. Analyzing the level of expression of an interferon-response gene, such as oligoadenylate synthase-1 (OAS1), interferon-stimulated gene-54 (ISG54), and guanylate-binding protein (GBP), in the transfected or transduced cells by northern blot or RT- PCR assays are commonly used.

Can there be any more convenient alternative method for checking interferon response? One potentially useful product could be HiTiter™ pre-packaged lentiviruses that would have a fluorescent protein (mTFP1, mWasabi, or the brightest FP in lanYFP) under the control of an ISRE (IFN-stimulated response element) or GAS (IFN gamma-activating sequence)*. This could be another group of Product-on-Demand type of reagents, meaning that we will have the design ready, but only to produce them upon ordering. This way the cost to us and the price to customers can be kept at minimum.

To read the wholr blog, click here.

Allele Granted Broad Patent on RNAi in China

Allele Biotechnology & Pharmaceuticals, a San Diego based private company with associate offices and laboratories in China and distribution channels in 30 countries, was granted a major landmark patent in China in the field of RNA interference (RNAi). The patent CN02828345.7, issued on January 20, 2010, covers compositions of DNA molecules that can be transcribed into RNAi-mediating RNA molecules, including the commonly used shRNA and miRNA-like designs. The patent also grants Allele Biotech rights to the process of introducing such DNA molecules into cells. To induce gene silencing by RNA interference, researchers often bring DNA molecules that encode interfering RNAs into cells via plasmid or viral vectors. The rights to use related technologies for the purposes of completely or partially abolishing gene functions through the mechanism of RNAi are granted to Allele Biotech.



Additional claims include methods of studying gene functions using DNA-encoded RNAi agents, or modifying gene expression profile by introducing gene expression-altering DNA molecules that will induce RNAi. The patent further protects the use of DNA-mediated RNAi in creating cell, animal models, and for curing human diseases. Allele Biotech intends to fully realize the value of this broad patent by providing opportunities to R&D centers, service providers, and reagent sellers to license at reasonable fees, so that this great technology will continue to be widely used and further developed through original research and investment.



For more views and comments by Allele Biotech and responses from others, please visit the Allele Blog on the same topic.



Website http://allelebiotech.com/blogs/

For further information: 858-587-6645, Fax 858-587-6692 RNAi@allelebiotech.com

Choosing siRNA, shRNA, and miRNA for Gene Silencing

http://allelebiotech.com/blogs/2010/02/choosing-sirna-shrna-and-mirna-for-gene-silencing/
...shRNA can be introduced by DNA plasmid, linear template, or packaged retroviral/lentiviral vectors. Using any form of DNA construct, except the PCR template format such as 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 [6, 7]. For the goals of this research, the most important advantage using shRNA can provide over siRNA is that it can be carried on a lentiviral vector and introduced into a wide variety of cells.

Similar to the comparison between siRNA versus shRNA, it is also difficult to rank the efficiency of shRNA versus miRNA from published data, partly due to different results from different experimental systems. There have been several reports that showed shRNA can cause significant cell toxicity, especially in vivo such as after injection into mouse brain. It was originally reasoned that highly efficient expression from Pol III promoters might overwhelm the cellular machinery that is needed to execute endogenous RNAi functions such as transporting miRNA from the nucleus to the cytoplasm. It was later found out that even using Pol III promoter to create miRNA could still mitigate the toxic effects of shRNA [8]. Since shRNA and miRNA are processed by endonuclease Dicer before being incorporated into RNA induced silencing complex (RISC), the exact identity of siRNAs produced from a given shRNA or miRNA targeting the same region on the mRNA are not known in most of the earlier studies. By designing shRNA and miRNA to give exactly the same processed siRNAs, Boudreau et al. showed that shRNA is actually more potent than miRNA in various systems [9].

New Product/Service of the Week (02-01-10 to 02-07-10): Lentrivirus retrovirus shRNA Packaging Services as low as under $900 per virus.

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For References see original post here please.

Effective Concentrations and Effectiveness of siRNA

RNA oligo is significantly more difficult to synthesize than DNA oligos, mainly because the efficiency of coupling each new ribonucleotide during RNA synthesis is a few fold lower than deoxyribonucleotide during DNA synthesis. Typically, there is an ~10% chance a DNA oligo of 21 bases will have a mutation (most frequently a deletion mutation); for an RNA oligo of 21 bases, as in an siRNA pair, such chance is much higher. Furthermore, after combining the sense and antisense siRNA strands, some RNA molecules will remain as single-stranded thereby not fitting for the RNAi apparatus.

RNA interference is a dose-sensitive process — specificity of gene silencing is meaningful only relative to the active concentration of siRNA used. When the concentration is too low, even the most effective siRNAs would fail to cause gene expression knockdown; when too high, non-specific effects will be duly observed. Therefore, it is essential that the concentrations of siRNAs are measured correctly. When doing so, one must consider not only what the apparent concentrations are by OD260 reading, but also whether the RNA strands are of full-length and whether only dsRNA molecules are counted. This issue might not affect data interpretation if appropriate controls are included in one set of RNAi experiments, but it could have significant influence on conclusions if data from different experiment sets or labs are compared or combined.

HPLC purification currently provides the best means to remove RNA molecules with deletions or remain single-stranded, however, the price tag added by most reagent providers for such treatment has been prohibiting because manufacturers either need to start synthesis at a much bigger scale to obtain promised amount, or they do not promise the delivery quantity at all. The phosphoramidites (oligo building blocks) for RNA synthesis can be 10 times or more expensive than for DNA. Some companies offer alternative purification methods such as a cartridge type device, but they can only remove salt and small impurities, not RNA oligos of shorter lengths accumulated at each cycle of amide coupling. The AllHPLC siRNAs within Allele’s RNAi product line, pre-validated or custom made, are uniformly HPLC purified with 5 OD or 12.5 nmol of double-stranded, annealed siRNA delivered. Allele passes to customers the cost savings from manufacturing our own RNA amidites and other reagents for oligo synthesis. The pre-validated HPLC purified double-stranded siRNA is offered today at $149/12.5 nmol.

Before purchasing siRNAs, even at a low cost of $29 per pair of HPLC purified control siRNA from Allele, researchers still need to consider how well their cells can be transfected. For hard-to-transfect cells, lentiviral vectors carrying a shRNA expressing cassette is often a better choice. To establish stable cell lines, plasmid vectors should be considered. For low cost target screening, the PCR format linear siRNA expression cassettes have advantages.