Marker Gene Monthly Newsletter   

November, 2006

Volume 6 , Number 11

© Copyright MGT, Inc., 2007.  Published by Marker Gene Technologies, Inc., The University of Oregon Riverfront Research Park, 1850 Millrace Drive, Eugene, Oregon 97403-1992 USA.  All rights reserved.  For information on the use or copying of the material contained in this document, please contact us at techservice@markergene.com.  Please see below for subscription information and updates.  This newsletter is labeled as an ADVERTISEMENT in accordance with the CAN-SPAM act of 2003, S.877 Public Law: 108-187.

Measuring siRNA Gene-Silencing in Mammalian Cells Using Marker Genes.

The use of small interfering RNA (siRNA) has renovated the field of gene-silencing in cultured mammalian cells.  The method involves the production of antisense RNA molecules, 21-23 bases in length, that will hybridize to a chosen homologous sequence of mRNA.  The silencing occurs by a process involving the ribonuclease-III nuclease, dicer, and is thought to be a natural silencing mechanism of exogenous transgenes from viral infection, or even a control mechanism for homologous genes in both plants and animals.  The resulting double-stranded RNA (dsRNAs) are highly unusual in the cell and are quickly processed by dicer, in association with a multimeric nuclease complex termed the RNA-induced silencing complex, which leads to degradation of the mRNA and effectual gene silencing.  Current methods of identifying potential siRNAs involve using computer algorithms to first select plausible candidate sequences, after which these synthesized RNAs are used to test knock-down in cells via Northern blot hybridization, quantitative reverse transcriptase PCR, Western blot or by immunofluorescence, if an antibody for the protein is available.  To circumvent the cost and the inconvenience in identifying a unique siRNA duplex that can quench target gene expression, researchers in the laboratory of Dr. Yi-Hsin Liu at the Center for Craniofacial Molecular Biology, at the University of Southern California, Los Angeles, CA have devised a modified luciferase expression vector system to test knockdown efficiency of selected siRNAs. They were able to demonstrate that this luciferase-based siRNA testing system can be used to evaluate the knockdown efficiency of a directly transfected siRNA duplex or an siRNA expressed from a lentiviral vector.  Basically, the method involves creating an in-frame fusion vector between the target gene and the luciferase gene in the plasmid, and then co-transfection with each target siRNA.  The siRNAs, if active, should shut down luciferase production as well as the target gene in the vector.  A pCMVb-lacZ expression vector was used as an internal control as were siRNAs for random sequences.  By performing dual luciferase/b-galactosidase assays, the gene-silencing of each siRNA could be determined, quickly and quantitatively.  The siRNA could be also adapted for use with siRNAs that were expressed via a lentiviral vector construct.  Knowing the quantitative efficiency of individual siRNAs will allow flexibility in performing dose-dependent gene silencing by this method.   This type of assay could also prove to be quite useful to evaluate the therapeutic potential of siRNAs.  Marker Gene provides several substrates for sensitive intracellular analysis of the reporter marker genes, including D-luciferin (M0237), the principle substrate for measuring luciferase activity, the MarkerGene^TM Live Cell Luciferase Assay Kit (M0626), D-Luciferin, ethyl ester (M0906), a more cell-permeant version of the native D-luciferin, as well as the MarkerGene^TM Chemiluminescent lacZ ß-Galactosidase Detection Kit (M0855) and the MarkerGene^TM in vivo lacZ ß-Galactosidase Intracellular Detection Kit (M0259).  For more information about these techniques, please visit our website or see the references below.

• Zhuang, F., Liu, Y.H., (2006) “Usefulmess of the luciferase reporter system to test the efficacy of siRNA.” Meth. Mol. Biol. 342:181-187.
• Bass, B.L., (2000) “Double-stranded RNA as a template for gene silencing” Cell 101:235-238.
• Hannon, G.J., Rossi, J.J., (2004) “Unlocking the potential of the human genome with RNA interference.” Nature 431:371-378.
• Du, Q., Thonberg, H., Wang, J., Wahlestedt, C., Liang Z., (2005) “A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites” Nucleic Acids Res. 33(5): 1671–1677.
• Whitehead Institute for Biomedical Research siRNA site: http://jura.wi.mit.edu/bioc/siRNAext/

Fluorescent Labeling of Oligonucleotides.

DNA microarrays allow the study of thousands of genes simultaneously, providing the ability to identify targets for metabolic regulation of gene activity.  In the case of cDNA arrays, the array itself is hybridized with the two different types of RNA or cDNA of interest (i.e. control and target) each of which must be labeled for identification.  One of the most common methods involves the use of fluorescent dye labeling (for example CY3, CY5, ROX, JOE, TAMRA or FAM) for microarray hybridization detection.  The dyes can be attached to the cDNA either by using incorporation of dye-conjugated nucleotide analogs during the reverse transcription process, or by incorporating aminoallyl-nucleotides and later reacting these with fluorescent NHS ester dyes.  A limitation of directly labeling with fluorescent nucleotides is that they are not the normal substrates for the DNA polymerase enzymes and since these nucleotides are often quite bulky, the efficiency of incorporation by PCR can be significantly lower than for the natural substrates. Also, fluorescent nucleotide analogs tend to be quite expensive.  The alternative is to use less inhibiting aminoallyl nucleotide (aaNTP) analogs that have a chemically reactive group to which a fluorescent dye may be later attached.  For example, aminoallyl-dUTP can be incorporated into cDNA which will then contain a primary amine to which an N-hydroxysuccinimidyl (NHS) ester reactive dye can be attached.  The effects of this labeling method on melting temperature, extinction coefficient and quantum yield, as a function of fluorophore density and linker arm length have been investigated and an optimal density of fluorophores on the DNA chain has been found to be at a spacing of greater than six bases/fluorophore label.  Higher densities were found to decrease both the fluorescence intensity, hybridization and the quantum yield of the labeled probe.  This is likely due to fluorophore-fluorophore or fluorophore-nucleotide base interactions that result in quenching or poor annealing.  The labeling density can be controlled by adjusting the amount of aa-dUTP in relation to the amount of dTTP used in the RT reaction.  Depending on the overall A/T content of the genome in question, the aaUTP concentration is adjusted.  For example, in yeast, the A/T content is about 60%, and a ratio of 2 aa-dUTP molecules to 3 dTTP molecules is recommended.  For the reverse transcription reaction (amplifying an RNA sample) a cocktail containing the reverse transcriptase enzyme, an oligo(dT)12-18-mer primer, Aminoallyl-dUTP (10 mM) and the nucleotides dATP, dCTP, dGTP, dTTP (100 mM each) works well.  Priming of the reverse transcription can also be done using random 15-mer oligonucleotides or an equal mix of both random 15-mer and oligo-dT.  If the probe is hybridized to an oligonucleotide array, it is better to use random primers or a mix of oligo-dT and random primers.  The typical method for NHS-ester fluorescent dye incorporation with amino modified oligos (C6-dT) obtained from cellular RNA or DNA is carried out in a sodium borate buffer at pH 8.5 (or 0.1 M Carbonate Buffer, pH 9) and purification can be achieved by ethanol precipitation followed by denaturing gel electrophoresis. The fluorescent dyes are dissolved at a high concentration in DMSO, split in aliquots and if not used immediately, kept frozen and in the dark at 4^oC (desiccated).  Marker Gene provides several of the fluorescent reactive NHS-ester dyes that can be used for these microarray analyses, including 5(6)-Carboxyfluorescein, NHS ester (FAM: M0984) and tetramethylrhodamine, NHS ester (TAMRA: M0972) as well as Biotin-X, SE (M0783) for use in labeling with biotin.  For more information about these techniques, please visit our website of see the references below. 

• Beier V., Bauer A., Baum M., Hoheisel J.D. (2004) “Fluorescent sample labeling for DNA microarray analyses.” Methods Mol Biol. 283:127-35.
• Richter A., Schwager C., Hentze S., Ansorge W., Hentze M.W., Muckenthaler M., (2002) “Comparison of fluorescent tag DNA labeling methods used for expression analysis by DNA microarrays.” Biotechniques. 33(3):620-8, 630.
• Randolph, J. B.,  Waggoner, A. S. (1997)  “Stability, specificity and fluorescence brightness of multiply-labeled fluorescent DNA probes.” Nucleic Acids Res 25(14), 2923-9.
• Brumbaugh, J. A., Middendorf, L. R., Grone, D. L., Ruth, J. L. (1988) “Continuous, online DNA sequencing using oligodeoxynucleotide primers with multiple fluorophores.” Proc Natl Acad Sci U S A 85(15), 5610-4.
• Hughes, T. R. et al. (2001) “Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer.” Nat Biotechnol 19(4), 342-7.
• De Freitas, J.M., Kim, J.H., Poynton, H., Su, T., Wintz, H, Fox, T., Holman, P., Loguinov, A. Keles, S. van der Laan, M. and Vulpe, C (2004) “Exploratory and Confirmatory Gene Expression Profiling of mac1delta.” J. Biol. Chem. Vol. 279:4450-4458.
• Enders, G., (2004) “Gene profiling--chances and challenges.” Acta Neurochir Suppl. 89: 9-13.

Digoxigenin versus Biotin Labeling.

Biotin and digoxigenin have

become widely used as labeling agents to detect cellular DNA or RNA.  However, emphasis has recently been placed on false-positive results that are sometimes obtained when biotin is used, especially in whole cell preparations, since endogenous biotin can sometimes interfere with specific signals.  D-Biotin is a small vitamin molecule (MW = 244), that binds with high affinity (kD 10^-15 M^-1) toavidin, a protein largely distributed in egg whites (MW =  70,000), or streptavidin.  These proteins can then be further conjugated to additional markers such as fluorescent dyes, enzymes like peroxidase, ferritin, or colloidal gold.  With the advent of biotin-labeled (d)UTP, the construction of biotinylated nucleic acids has also become possible.  However, problems of nonspecific binding have been reported, which can often be attributed to non-specific binding of avidin or streptavidin presumably caused by carbohydrate interactions or ionic interactions with anionic cell surfaces (pI _(avidin) = 10) and partially explain nonspecific binding of avidin conjugates.  Nevertheless, biotin labeling has become a standard technique for DNA/RNA labeling, in microplate assay systems (Affymetrix, etc.)

   Digoxigenin presents itself as a good alternative to the biotin-avidin system, mainly because it typically shows very low non-specific binding due to its natural presence only in Digitalis plants as a secondary metabolite.  Detection of hybridized digoxigenin-labeled probes is mediated by high-affinity anti-digoxigenin antibodies conjugated to either alkaline phosphatase, peroxidase, fluorescein, rhodamine or colloidal gold or revealed by a secondary antibody that is similarly labeled. The use of unconjugated anti-digoxigenin antibodies with conjugated secondary antibodies seems to enhance the detected signal.  Although the use of digoxigenin can be somewhat more expensive, detection limits have routinely been found to be slightly better than when using biotin labeling.  Marker Gene provides several key labeling reagents for these assays including Biotin, Succinimidyl Ester (M0785), Biotin-X, SE (M0783), Biotin-Dextran (70,000 MW) (M0788), Avidin Sulforhodamine 101 Conjugate (Texas Red^TM equivalent) (M1124) and the MarkerGene^TM Biotin-X Antibody/Protein Labeling Kit (M1138).  For more information about these techniques, please visit our website or see the references below.

• Zhu T., Chang S.H., Gil P., (2006) “Target preparation for DNA microarray hybridization.” Methods Mol. Biol. 323: 349-57.
• Hu Z.,  Zhang A., Storz G., Gottesman S., Leppla S.H., (2006) “An antibody-based microarray assay for small RNA detection.” Nucleic Acids Res. 34(7): e52.
• Green NM (1975) “Avidin”  Adv. Protein Chem. 29:85-133. Brigati D., Myerson D., LearyJ., Spalholz B., Travis S., Fong C., Hsiung C., Ward D., (1983) “Detection of viral genomes in cultured cells and paraffin-embedded tissue sections using biotin-labeled hybridization probes.” Virology 126:32-50.
• Kirkeby S., Moe D., Bog-Hansen T.C., Van Noorden C.J.F., (1993) “Biotin carboxylases in mitochondria and the cytosol from skeletal and cardiac muscle as detected by avidin binding.” Histochemistry 100:415-421.

Cellulase Activity Measurements.

Cellulases are a family of three enzymes:  ß-Glucosidases, endoglucanases, and exoglucanases.  These enzymes cleave the ß-1,4-D-glycosidic bonds that link the glucose units comprising cellulose.  In addition to being produced by plants, cellulase activity is found in many fungi and bacteria, including some plant pathogens.  No mammalian cells are known to produce cellulase, however, cellulolytic activity is often carried out in animals by symbiotic bacteria.  The study of cellulase activity has many applications in plant molecular biology, agriculture, and manufacturing.  Cellulase is also becoming important in the development of alternative fuel sources, as glucose obtained from cellulose hydrolysis is easily fermented into ethanol. Marker Gene Technologies is currently developing both colorimetric and fluorometric substrates, as well as versatile kits, for the assay of cellulase activity, including the currently available chromogenic substrate 2-Chloro-4-Nitrophenyl-b-D-Cellobioside (M0637), with absorbance at 405 nm after enzyme activity.  For more information regarding cellulase assays, please visit our website or see the references below.   

• Zhang Q., Bai G., Yang W., Li H., Xiong H. (2006).  “Pathogenic cellulase assay of pine wilt disease and immunological localization.’ Biosci. Biotechnol. Biochem. 70(11): 2727-32
• Nakata T., Miyafuji H., Saka S.(2006) “Bioethanol from cellulose with supercritical water treatment followed by enzymatic hydrolysis.” Appl. Biochem. Biotechnol. 129-132: 476-85
  
• Shani Z., Dekel M., Roiz L., Horowitz M., Kolosovski N., Lapidot L., Alkan S., Koltai H., Tsabary G., Goren R., Shoseyov O.  (2006) “Expression of endo-1,4-β-glucanase (cel1) in Arabidopsis thaliana is associated with plant growth, xylem development and cell wall thickening.” Plant Cell Rep, 25: 1067–1074
Han S.J., Yoo Y.J., Kang H.S.  (1995) “Characterization of a Bifunctional Cellulase and Its Structural Gene.”  J. Biochem, 270(43):  26012-26019.

• Thayer D.W.  (1978) “Carboxymethylcellulase produced by facultative bacteria from the hind-gut of the termite Reticulitermes hesperus.”  Journal of General Microbiology, 106(1) 13-8

Compare Our Quality. 

Marker Gene strives to offer our customers products of the highest quality and at the best possible prices.  Our years of experience allow us to provide timely products for less cost to you.  See our latest Price Comparison Chart that compares our prices with those from several alternate sources, to see if you can save money by switching to Marker Gene (http://www.markergene.com/crossref.htm).  Or visit our website at www.markergene.com and click on the link “COMPARE”.  We think you will appreciate our efforts to keep costs low and maintain excellent quality of our products for your research.  For more information about any of our products, simply telephone us toll free at 1-888-218-4062 or contact us by e-mail at techservice@markergene.com.  We will be happy to send you more about our products and their specifications.

CONTRACT  RESEARCH@markergene.com

Marker Gene Technologies, Inc. has the expertise to perform contract research with you on your project. We have worked with many biotechnology and pharmaceutical companies on successful, proprietary and patented projects.

Contract Research and Development Capabilities in the following areas:

• Established in 1993 at the University of Oregon Riverfront Research Park.
• Screening Assay Development for HTS and uHTS
• Chemical and Cellular Assays – High-Content Screening.
• DNA/RNA (genomics) and protein (proteomics) labeling and assay development.
• Pharmaceutical Intermediates - design, synthesis, and in vitro testing in mammalian cell culture.
• Specializing in Carbohydrate, Lipid, Peptide, and Nucleic Acid Chemistries.
• Fully equipped laboratories (Biochemistry, Chemical Synthesis, Tissue Culture, Analytical).
• Confidentiality, help in patent preparation and filings.

Contact us by telephone at (888) 218-4062 or (541) 342-3760 or FAX us at (541) 342-1960 or you can write to us at  Contract Research, Marker Gene Technologies, Inc., 1850 Millrace Drive, Eugene, Oregon 97403-1992 or contact us by e-mail at: techservice@markergene.com

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