Choosing siRNA, shRNA, and miRNA for Gene Silencing
RNAi refers to dsRNA-induced gene silencing, a cellular process that degrades RNA homologous to one strand of the dsRNA [1, 2]. The intermediates of long dsRNA-initiated RNAi are double-stranded small interfering RNAs (siRNA), typically 21-23 nucleotide (nt) long. The siRNAs, when introduced into cells, can be used to silence genes in mammalian systems where long dsRNAs prompt protein kinase R (PKR), RNase L, and interferon activities that result in non-specific RNA degradation and general shutdown of protein synthesis [3]. siRNAs can either be chemically synthesized then directly transfected into cells or can be generated inside the cell by introducing vectors that express short-hairpin RNA (shRNA) precursors of siRNAs. The process of shRNA into functional siRNA involves cellular RNAi machinery that naturally process genome encoded microRNAs (miRNA) that are responsible for cellular regulation of gene expression by modulating mRNA stability, translation, and chromatin structures [4].
Chemically synthesized siRNA is the simplest format for 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 reachable 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, typically with gene silencing activities peaking around 24 hours, and diminishing within 48 hours [5]. Chemical synthesis of siRNA, which is a service Allele Biotech and Orbigen (now merged under the Allele brand) pioneered and still provides, is expensive on a per transfection basis relative to DNA vector based reagents.
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.
Promotion of the week: Get mouse tail lysis buffer, human blood genotyping buffer, or DNA purification kit, and get Allele Biotech’s superior PCR MasterMix for free.
1. Fire, A., S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver, and C.C. Mello, Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998. 391(6669): p. 806-11.
2. Hannon, G.J., RNA interference. Nature, 2002. 418(6894): p. 244-51.
3. McManus, M.T. and P.A. Sharp, Gene silencing in mammals by small interfering RNAs. Nat Rev Genet, 2002. 3(10): p. 737-47.
4. Hutvagner, G. and P.D. Zamore, A microRNA in a multiple-turnover RNAi enzyme complex. Science, 2002. 297(5589): p. 2056-60.
5. Rao, D.D., J.S. Vorhies, N. Senzer, and J. Nemunaitis, siRNA vs. shRNA: similarities and differences. Adv Drug Deliv Rev, 2009. 61(9): p. 746-59.
6. Vlassov, A.V., B. Korba, K. Farrar, S. Mukerjee, A.A. Seyhan, H. Ilves, R.L. Kaspar, D. Leake, S.A. Kazakov, and B.H. Johnston, shRNAs targeting hepatitis C: effects of sequence and structural features, and comparision with siRNA. Oligonucleotides, 2007. 17(2): p. 223-36.
7. Siolas, D., C. Lerner, J. Burchard, W. Ge, P.S. Linsley, P.J. Paddison, G.J. Hannon, and M.A. Cleary, Synthetic shRNAs as potent RNAi triggers. Nat Biotechnol, 2005. 23(2): p. 227-31.
8. McBride, J.L., R.L. Boudreau, S.Q. Harper, P.D. Staber, A.M. Monteys, I. Martins, B.L. Gilmore, H. Burstein, R.W. Peluso, B. Polisky, B.J. Carter, and B.L. Davidson, Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: implications for the therapeutic development of RNAi. Proc Natl Acad Sci U S A, 2008. 105(15): p. 5868-73.
9. Boudreau, R.L., A.M. Monteys, and B.L. Davidson, Minimizing variables among hairpin-based RNAi vectors reveals the potency of shRNAs. Rna, 2008. 14(9): p. 1834-44.
How We Give Back in 2010
This year has started off great for Allele. Every week our shipping volume is increasing, we have an arsenal of new products in the pipeline, we are the sole provider of oligos on the University of California San Diego’s central online purchasing site, and every day we are taking even more action to cut the waste and increase productivity in order to give our customers the very best at a reasonable cost. Our company is maturing nicely in its 10th year!
Lately we have really been focusing on giving back to our customers who I, on a daily basis, am impressed with. Most of all, I am impressed with how good of a relationship we have. I have several customers who I am on the phone with regularly and genuinely glad to hear from. We have fun with our friends on facebook, giving advice on molecular biology techniques and even swapping recipes.
So far this year we have designed three new ways to reward our customers:
There is our big 10 Year Anniversay Oligo Giveaway where we will give away a free month of oligos to one facebook friend or fan. DNA and RNA oligos were Allele’s very first product and we knew we wanted to have a big prize to reward to celebrate not only 10 years in business but also surviving through what is the worst economic time in decades. It only made sense that the giveaway should be oligos. Most importantly, this is also where our longest customer relationships exist; within our oligo customer base. Personally, I cannot wait for the winner to be announced. I am probably more excited about it than most of the contestants!
This last January we also launched our Allele Biotech Rewards Program designed by our Business Development Manager Eugene Pahk. We want to make it more affordable for all Allele customers to purchase Allele Biotech Products (we have over 1000). Allele Rewards are easy to earn and more importantly, easy to redeem. We do not make it complicated, rewards do not expire, and they can be used on a number of common molecular biology research tools. Just another way to give back to the people who keep us in business and support our research.
Our latest tribute to our patrons is especially designed for all the younger, tech savvy (and usually desperately in need of money) grad students! We will award $100 cash to anyone who submits an Allele product or service relevant picture to us for use on our homepage. We are tired of the current photos we have now and instead of just purchasing some (which we could do for a really low cost) we decided to get the force of our creative and intelligent supporters to not only have some fun but to get a different perspective on what the Allele brand means to people.
On top of the new programs and promotions, every week we make a promise to have one item or service on sale; sometimes at the direct behest of the researchers themselves. Our weekly promotions are announced via our social networking sites (facebook, twitter, Linkedin, myspace, and our Allele Biotech Blog). We want to have as many avenues as possible for customers to be involved with Allele Biotech so we can succeed in this business for decades to come while giving back to our customers the entire way.
Become a friend, fan, follower, contact, or bookmark our blog site today to get the most of what we give!
Monitoring the Undifferentiated Stage of Stem Cells—the Pluripotency Markers
Human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells promise to serve as an unlimited source for transplantation or tissue-specific differentiation. However, obtaining and maintaining stem cells are very difficult tasks for multiple reasons. For instance, most stem cell lines tend to spontaneously differentiate in culture, and even if the cells form stem cell-like colonies, they may be of a heterogeneous population.
To identify pluripotency of stem cells, expression of stem cell-specific marker genes (i.e. Oct-3/4, Sox2, Nanog, Rex-1) is monitored by RT-PCR. Alkaline phosphatase activity and methylation profiles of promoters of pluripotency-relevant genes are often analyzed as well. Compared to murine cells, it is noticeably more difficult to obtain human iPSCs, of which stem cell-like colonies sometimes turn out not to be pluripotent cells. We highly recommend testing iPSCs, especially human iPSCs, with antibodies against stage-specific embryonic antigens such as SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81.
However, all of these methods require cell destruction or fixation for analysis, therefore, are inconvenient and costly. Furthermore, many studies using ES or iPS cells involve differentiation of stem cells into different lineages, a method for observing live cells to know their undifferentiation/differentiation stages would be very helpful. There have been a number of publications using murine Oct-4, Nanog, and Rex-1 promoter driven fluorescent proteins as markers for pluripotency tests [1-3]. Allele Biotech provides, under its iPS product line, packaged and validated lentiviral particles that would insert these 3 promoter-FP reporters into the stem cells. Although currently these promoters are of mouse sequences, their use in human stem cells have been reported.
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New product of the week 01-25-10 to 01-31-10:
All-In-One-Vector: Human OSKM Lentiviral Paticles, with Oct-4, Sox-2, Klf, and c-Myc all expressed from a single virus, ready-to-use.
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Promotion of the week:
human iPS cell detection primer set, the same as the landmark Yamanaka paper [4] on creating human iPS for the first time.
1. Da Yong WU, Zhen YAO (2005). Isolation and characterization of the murine Nanog gene promoter. Cell Research, 15 (5): 317–324.
2. Rachel Eiges, Maya Schuldiner..et.al (2001). Establishment of human embryonic stem cell?transfected clones carrying a marker for undifferentiated cell. Current Biology 11: 514–518.
3. Guangjin Pan, Jun Li, Yali Zhou, Hui Zheng, and Duanqing pei (2006). A negative feedback loop of transcription factors that control stem cell pluripotency and self?renewal. ASEB Journal 20: E1094? E1102
4. Takahashi et al, Induction of Pluripotent Stem Cell from Adult Human Fibroblasts by Defined Factors (2007). Cell 131, 861-872
Submit Your Pictures and Enter to Win $100 in Cash
Allele Biotech is holding another fun contest to give back to our loyal supporters and everyone has the chance to win $100 in cash! We are looking for interesting and relevant pictures, illustrations, and computer generated art that pertains to any Allele Biotech Product or service to replace the flashes on our homepage. If it is chosen to be used we will give you $100 cash!
Check out our technical data sheets, read our past blogs, and even use your own lab experience and impressions with Allele Biotech Products to come up with your entry. There are over 1000 products to choose from so everyone should have lots of inspiration!
All you have to do to enter is submit your picture to our facebook inbox! The picture has to be of your own creation and/or one to which you own exclusive rights. Response time and prizes will take about a week.
Let your creative juices flow and you could get $100 in return. Your picture could be funny, serious, or even super nerdy!
Used to promote our Sapphire(TM) Baculovirus Expression Service
All photos, illustrations, and computer generated graphics (aka. “the picture”) submitted must be lawfully owned by entrants who submitted them. Pictures that are not chosen will not be used in any way by Allele Biotech. Winners may collect their prize via cash, check, or a one-time Allele credit of $100 good toward any Allele Biotech purchase. Allele Biotech has full rights to pictures selected for use in Allele advertising and winners must forfeit any future rights to the picture. Contest to run indefinitely and to be terminated at any time.
New Product for 1-18-10 to 1-24-10 High Throughput DNA Oligos!
Need a plate of high quality oligos fast and for a great price? Allele’s newest product can help…
Introducing High Throughput DNA Oligos!
Anywhere from 48 – 96 desalt oligos per plate
Oligos 20 – 50 bases long
Available in two synthesis scales:
25 nmol scale only 13 cents per base
50 nmol scale only 17 cents per base
Every oligo is strictly controlled for quality:
Oligo quality is verified using MALDI-TOF mass spectrometry or Electrospray Ionization Mass Spectrometry.
Shipped or hand delivered in 5 days!
Oligos can be provided normalized at a specific concentration with fixed or variable volumes. Concentration options depend on the oligo length, purification, and scale of synthesis being ordered.
No minimum number of plates per order required!
To order, simply email your oligo sequences and names, indicate 25 nmol or 50 nmol, concentration preference in excel or notepad format to oligo@allelebiotech.com A template will be posted online here soon, please check back.
Allele…Continuing to introduce cost efficiency to research!
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