Lab Skills You Stopped Being Proud Of

Molecular biologists who were in graduate school in the 90’s learned how to isolate plasmid DNA from E. coli cultures by a method called “boil-prep” during their first lab rotation. This process involved mixing the bacterial cell pellet in a little bit of detergent, salt and sucrose, dabbed with some fresh lysozyme, and then you are ready to cook, literally! Bacterial cell membranes are disrupted by boiling this soup in a beaker of water over a Bunsen Burner for one minute, and the debris (containing the broken cell membrane and attached chromosomal DNA) is collected by centrifugation in a microfuge at top speed for 10 minutes. Then comes the step that differentiates a true master of lab skills versus a rotation student—if you knew just the right amount of bacterial culture to begin with and handled the E coli pellet by the right techniques, a skillful lab person could collect nearly all the liquid without disturbing the pellet. Pouring out the plasmid-containing supernatant without dislodging the goo on the side/bottom of the tube was such a desirable skill that would not only give you your plasmid but also give you admiration from fellow lab members. That is, of course, if you were doing it before the mid-90’s, because after the introduction of miniprep spin columns by Qiagen, nobody, even the true masters of boil-preps (or its contemporary alkali prep that also involves pelleting by centrifugation and careful removal of tiny volume of liquids surrounding small pellets) would be showing off those skills any longer.

It is actually never easy or fun to collect liquid surrounding small amount of beads or pellets as you always have to struggle to remove as much liquid as possible while trying not to lose any of the beads

Some of the old-timers used to also be very proud of being able to pour a “sequencing gel” (a very thin ~40 cm x 30 cm polyacrylamide gel). I still remember the first time I reported to the second rotation lab at USC. After describing the lab research, the PI showed me around the lab and complained how “Sarah destroyed all my sequencing gel plates”. But consider this, in order to avoid any greasy spot on either plate, you needed to wash both of them fanatically if not religiously. Why? You would have just about a minute’s time to pour non-polymerized acrylamide without leaking from the sides or bubbles forming anywhere in the DNA running lanes, and then inserting a pair of paper-thin combs, all at a speed quicker than TEMED/AP-catalyzed acrylamide polymerization. Good thing that after capillary sequencing was invented, we all happily retired our sequencing-gel pouring skills with a collective sigh of relief.

Technology will always move forward, so will the skills lab researchers will be required to perfect. Using a spin column is very much a “skill-less” technique in contrast to collecting pellets and washing beads after centrifugation, but when there is a choice, people will chose the method that requires “less skills”, such as the spin-column format as the preferred platform for the new FP-nAb™ products.

BTW, like to have information on the spin column kit? Here it is: http://www.allelebiotech.com/gfp-nab-agarose-spin-kit-20-reactions/

Tags: agarose beads, camelid antibody, capture reagent, co-IP 101, GFP, gfp affinity tag, gfp antibody, GFP co-IP, GFP-nAb, magnetic beads, mNeonGreen-nAb, nano antibodies, nanobodies, polyacrylamide beads, RFP, spin column, Trap, VHH domain

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.

Currently Trendy Product Line: Camelid antibody group against fluorescent proteins as precipitation tag for co-IP (replacing formerly GFP-Trap line)–GFP-nAb, promotion ongoing now.

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.

Tags: alpaca antibodies, camelid antibody, gene knockdown, gene silencing, GFP-Trap, lentivirus packaging, miRNA, nanobodies, packaging systems, RFP-Trap, RNAi, shRNA, shRNA packaging, siRNA, VHH, virus packaging service