Validation of short interfering RNA knockdowns by quantitative real-time PCR

Methods in Molecular Biology

Volumn 353, Issue , 2007, Pages 177-203

  Tuzmen, Sukru ^a   Kiefer, Jeff ^a   Mousses, Spyro ^a  

^a NONE

Author keywords

Dicer; Gene expression; Gene knockdown; Gene quantification; Gene silencing; Housekeeping genes; Nonradioactive analysis; Quantitative real time PCR; Reference genes; Relative quantification; RISC; RNA; RNAi; siRNA; Transcription

Indexed keywords

MAMMALIA;

COMPLEMENTARY DNA; DNA; RNA; SMALL INTERFERING RNA;

ARTICLE; BIOSYNTHESIS; BREAST TUMOR; FEMALE; GENE EXPRESSION; GENETIC TRANSFECTION; GENETICS; HELA CELL; HUMAN; ISOLATION AND PURIFICATION; METHODOLOGY; ONCOGENE; PHENOTYPE; POLYMERASE CHAIN REACTION; RNA INTERFERENCE; STATISTICS; TUMOR CELL LINE; TUMOR SUPPRESSOR GENE; VALIDATION STUDY;

BREAST NEOPLASMS; CELL LINE, TUMOR; DNA, COMPLEMENTARY; DNA, NEOPLASM; FEMALE; GENE EXPRESSION; GENES, TUMOR SUPPRESSOR; HELA CELLS; HUMANS; ONCOGENES; PHENOTYPE; POLYMERASE CHAIN REACTION; RNA INTERFERENCE; RNA, NEOPLASM; RNA, SMALL INTERFERING; TRANSFECTION;

EID: 34247254427     PISSN: 10643745     EISSN: None     Source Type: Book Series    
DOI: 10.1385/1-59745-229-7:177     Document Type: Article

References (40)

1. Hannon, G. J. (2002) RNA interference. Nature 418, 244-251.
2. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494-498.
3. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., and Mello, C. C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811.
4. Hemann, M. T., Fridman, J. S., Zilfou, J. T., et al. (2003) An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo. Nat. Genet. 33, 396-400.
5. Carmell, M. A., Zhang, L., Conklin, D. S., Hannon, G. J., and Rosenquist, T. A. (2003) Germline transmission of RNAi in mice. Nat. Struct. Biol. 10, 91-92.
6. Tiscornia, G., Singer, O., Ikawa, M., and Verma, I. M. (2003) A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA. Proc. Natl. Acad. Sci. USA 100, 1844-1848.
7. Tuschl, T. (2001) RNA interference and small interfering RNAs. Chembiochem. 2, 239-245.
8. Caplen, N. J. (2003) RNAi as a gene therapy approach. Expert. Opin. Biol. Ther. 3, 575-586.
9. Wilda, M., Fuchs, U., Wossmann, W., and Borkhardt, A. (2002) Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi). Oncogene 21, 5716-5724.
10. Brummelkamp, T. R., Bernards, R., and Agami, R. (2002) Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2, 243-247.
11. Scherr, M., Battmer, K., Winkler, T., Heidenreich, O., Ganser, A., and Eder, M. (2003) Specific inhibition of bcr-abl gene expression by small interfering RNA. Blood 101, 1566-1569.
12. Kamath, R. S. and Ahringer, J. (2003) Genome-wide RNAi screening in Caenorhabditis elegans. Methods 30, 313-321.
13. Pothof, J., van Haaften, G., Thyssen, K., et al. (2003) Identification of genes that protect the C. elegans genome against mutations by genome-wide RNAi. Genes Dev. 17, 443-448.
14. Lum, L., Yao, S., Mozer, B., et al. (2003) Identification of Hedgehog pathway components by RNAi in Drosophila cultured cells. Science 299, 2039-2045.
15. Hammond, S. M., Bernstein, E., Beach, D., and Hannon, G. J. (2000) An RNAdirected nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404, 293-296.
16. Hammond, L. A., Davidson, K., Lawrence, R., et al. (2001) Exploring the mechanisms of action of FB642 at the cellular level. J. Cancer Res. Clin. Oncol. 127, 301-313.
17. Bernstein, E., Caudy, A. A., Hammond, S. M., and Hannon, G. J. (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363-366.
18. Azorsa, O.D., Mousses, S., Caplen, J. N. (2004) Gene silencing through RNA interfererence: Potential for therapeutics and functional genomics. Letters in Peptide Science 10, 361-372.
19. Bustin, S. A. (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J. Mol. Endocrinol. 29, 23-39.
20. Schmittgen, T. D. (2001) Real-time quantitative PCR. Methods 25, 383-385.
21. _ΔΔCT Method. Methods 25, 402-408.
22. Ginzinger, D. G. (2002) Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp. Hematol. 30, 503-512.
23. Schmittgen, T. D., Zakrajsek, B. A., Mills, A. G., Gom, V., Singer, M. J., and Reed, M. W. (2000) Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods. Anal. Biochem. 285, 194-204.
24. Schmittgen, T. D. and Zakrajsek, B. A. (2000) Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J. Biochem. Biophys. Methods 46, 69-81.
25. Chen, C. Y and Shyu, A. B. (1994) Selective degradation of early-response-gene mRNAs: functional analyses of sequence features of the AU-rich elements. Mol. Cell. Biol. 14, 8471-8482.
26. Iyer, V. R., Eisen, M. B., Ross, D. T., et al. (1999) The transcriptional program in the response of human fibroblasts to serum. Science 283, 83-87.
27. Giulietti, A., Overbergh, L., Valckx, D., Decallonne, B., Bouillon, R., and Mathieu, C. (2001) An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods 25, 386-401.
28. Niesters, H. G. (2001) Quantitation of viral load using real-time amplification techniques. Methods 25, 419-429.
29. Longo, M. C., Berninger, M. S., and Hartley, J. L. (1990) Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene 93, 125-128.
30. Varshney, U., Hutcheon, T., and van de Sande, J. H. (1988) Sequence analysis, expression, and conservation of Escherichia coli uracil DNA glycosylase and its gene (ung). J. Biol. Chem. 263, 7776-7784.
31. Lindahl, T., Ljungquist, S., Siegelt, W., Nyberg, B., and Sperens, B. (1977) DNA N-glycosidases: properties of uracil-DNA glycosidase from Escherichia coli. J. Biol. Chem. 252, 3286-3294.
32. Erlich, H. A., Gelfand, D., and Sninsky, J. J. (1991) Recent advances in the polymerase chain reaction. Science 252, 1643-1651.
33. Wilfinger, W. W., Mackey, K., and Chomczynski, P. (1997) Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Biotechniques 22, 474-476, 478-481.
34. Heid, C. A., Stevens, J., Livak, K. J., and Williams, P. M. (1996) Real time quantitative PCR. Genome Res. 6, 986-994.
35. Lakowicz, J. R. and Keating, S. (1983) Binding of an indole derivative to micelles as quantified by phase-sensitive detection of fluorescence. J. Biol. Chem. 258, 5519-5524.
36. Thellin, O., Zorzi, W., Lakaye, B., et al. (1999) Housekeeping genes as internal standards: use and limits. J. Biotechnol. 75, 291-295.
37. Vandesompele, J., De Prêter, K., Pattyn, F., et al. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, RESEARCH0034.1-0034.11.
38. Mullis, K. B. (1990) Target amplification for DNA analysis by the polymerase chain reaction. Ann. Biol. Clin. (Paris) 48, 579-582.
39. Walker, N. J. (2002) Tech.Sight. A technique whose time has come. Science 296, 557-559.
40. Pfaffl, M. W. (2001) A new mathematical model for relative quantification in realtime RT-PCR. Nucleic Acids Res. 29, e45.