5.4 对照设置：每个引物都要设无模板对照，阳性对照以及污染对照。
6.关于杂交探针的评价：在使用杂交探针进行实验时，必须注意防止探针－引物二聚体的形成和其本身在反应过程中的延伸。引物－探针二聚体的形成，主要是因为探针可与引物的3’末端杂交，其形成以后，会致使此二聚体扩增，从而同目的基因竞争反应的原料，致反应的效率下降。探针其本身能同目的基因相结合，且其解链温度高于引物，所以它可能作为引物而引发延伸反应，为了防止发生这种现象，通常是将其3’末端完全磷酸化，使之不能延伸，若此磷酸化不完全或是没有磷酸化，就会产生目的基因的副产品，从而干扰实验结果。鉴于以上这两点，所以应对探针精心设计，并将其末端完全磷酸化。
目前的实时定量PCR技术进展迅速，本文是结合本实验室的工作经验以及文献资料写成的，文中提到的一系列方案、实验材料和仪器设备都有其各自的优缺点，也有各自的使用范围，不能一概而论。所以不论研究者想进行何种研究，都应根据自己的情况首先找到适合自己研究目的的条件，本文仅供有意于运用实时定量PCR进行研究的人员参考。
参考文献：
1. Saiki, R.K., S. Scharf, F. Faloona, K.B. Mullis, G.T. Horn, H.A. Erlich and N. Arnheim. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985. 230:1350-1354.
2. Montogomery, R.A, and Dallman, M.J 1997 Cytokine 9,717-726.
3. Wall SJ, Edward DR. Quantitative reverse transcription-polymerase chain reaction (TR-PCR): A comparison of prime-dropping, competitive, and real-time RT-PCRs. Anal Biochem. 2002 Jan 15;300(2):269-73.
4. Schmittgen TD. Real-time quantitative PCR. Methods. 2001 Dec;25(4):383-5.
5. Willard M, Freeman, Stephen J. Walker and Kent E. Vrana. Quantitative RT-PCR: Pitfall and Potential. Bio Techniques 1999 Jan 26(1):112-125.
6. Giulietti A, Overbergh L,Valckx D,Decallonne B, Bouillon R, Mathieu C. An overview of real-time quantitative PCR: application to quantify cytokine gene expression. Methods 2001 Dec;25(4):386-401.
7. Rajeevan MS, Ranamukhaarachchi DG,Vernon SD, Unger ER. Use of real-time quantitative PCR to validate the results of cDNA array and differential display PCR technologies. Methods Dec 25(4):443-51.
8. Livark K.J., and Schmittgen T.D. 2001 Methods 25, 402-408.
9. Kawakami K, Brabender J, Lord R, Groshen S. 2000 J.Natl. Cancer Inst. 92,1805-1811.
10. Met Ulrich L., and Hans K. Real-time PCR analysis of DNA and RNA extracted from formalin –fixed and paraffin-embedded biopsies. 2001 Methods 25,409-418.
11. Leone G., van Schijndel H., van Gemen B., Kramer F.R., and Sxhoen C.D.1998 Nucleic Acids Res. 28,655-661.
12. Wittwer CT, Ririe KM, Andrew RV, David DA, Gundry RA and Balis UJ. 1997 Biotechniques 22 178-183.
13. Killgore GE, Holloway B and Tenover F. 2000 J. Clin. Microbiol.38 2516-2519.
14. Martell M, Gomez J, Esteban JI, Sauleda S, Quer J, Cabot B, Esteban R and Guardia J. 1999 J. Clin. Microbiol. 37,327-332.
15. Oleksiewicz M.B., Donaldson A.I., and Alexandersen S. 2001 J. Virol.Methods 92,23-35.
16. Van Trappen P.O., Gyselman V.G., Lowe D.G. and Rany A. 2001 Lacent 357,15-21.
17. Overbergh L., Valckx D, Waer M, and Mathieu C. 1999 Cytokine 11, 305-312.
18. Tyagi S, and Kramer F.R.. 1996 Nat .Biotechnol. 14,303-308.
19. Stryer L. 1978 Annu. Rev. Biochem. 47,819-846.
20. Gardullo R.A., Agrawal S., Flores C., Zamecnick P.C., and Wolf D.E., 1998 Proc. Natl. Acad. Sci. USA 85 8790-8794.
21. Bonnet G, Tyagi S, Libchaber A, and Kramer F.R .1999 Proc. Natl. Acad. Sci. USA 96,6171-6176.
22. Kaboev O.K., Luckkina L.A., Tretiakov A.N., and Bahrmand A.R. 2000 Nucl Acids Res. 28 e94.
23. Smit M.L, Giesenford B.A.J., Vet J.A.M., Trijbels F.J.M., and Blom H.J. 2001 Clin. Chem.47,739-744.
24. Durand R., Eslahpazire J., Jafari S., Delabre J.F., Mamorat-Khuong A., di Piazza J.P., and Le Bras J.2000 Antimicrob.Agents Chemother.44 3461-3464.
25. Fortin N.Y., Mulchandani A.,and Chen W.2001 Anal.Biochem 289,281-288.
26. Chen W., Martinez G., and Mulchadani A.2000 Anal.Biochem 280 160-172.
27. Lewin S.R., Vesanen M., Kostrikis L., Hurley A., Duran M., Zhang L., Ho D.D., and Markowitz M. 1999 J.Virol. 73, 6099-6103.
28. Pierce K.E., Rice J.E., Sanchez J.A., Brenner C., and Wangh L.J. 2000 Mol. Hum. Reprod. 6,1155-1164.
29. Vet J.A.M., Majithia A.R., Marras S.A.E., Tyagi S., Dube S., Poiesz B J., and Kramer F.r.,1999 Proc. Natl. Acad. Sci. USA 96,6394-6399.
30. Whitcombe D., Theaker J., Guy S.P., Brown T., and Little S 1999 Nature Biotechnol. 17 804-807.
31. Thelwell N., Millington S., Sollinas A., Booth J., and Brown T. 2000 Nucl Acids Res. 28,3752-3761.
32. Bernard P.S, and Wittwer C.T. 2000 Clin chem. 46 147-148.
33. Bellin T., Pulz M., Matussek A., Hemphen H.G., and Gunzer F.2001 J. Clin. Microb. 39 370-374.
34. Schalasta G., Eggers M., Schmid M., and Enders G. 2000 J. Clin. Virol. 19 175-185.
35. Simpson D.A.C., Feeney S., Boyle C., and Stitt A.W. 2000 Mol. Vis. 6, 178-183.
36. Ririe K.M., Rasmussen R.P., and Wittwer C.T., 1997 Anal. Biochem 245,154-160.
37. Kellogg D.E., Rybalkin I., Chen S., Mukhamedova N., Vlasik T., Siebert P.D., and Chenchick A. 1994 Biotechniques 16, 1134-1137.
38. de Baar M.P., van Dooren M.W., de Rooij D., Bakker M., van Gemen B., Goudsmit J., and de Ronde. 2001 J. Clin Microbiol. 39, 1378-1384.
39. Bustin S.A., 2000 J. Mol. Endocrinol. 25,169-193.
40. Martorana A.M., Zheng G., Springall F., Iland H.I., O’Grady and Lyons J.G. Absolute quantitation of Specific mRNA in cell and tissue samples by comparative PCR. 1999 Biotechniques 27,136-144.
41. Raeymaekers L. Basic principle of quantitative PCR. 2000 Mol. Biotech. 15, 115-122.
42. Zimmermann K., and Mannhalter J.W. Technical aspect of quantitative competitive PCR. 1996 BioTechniques 21,268-279.
43. Sarkar G., and M.E. Bolander 1994 The “looped oligo” method for generating reference molecules for quantitative PCR. Biotechniques 17,846-866.
44. Kohsaka H., A. Tanigushi, D.D. Richman and D.A. Carson . 1993 Microtiter format gene quantification by xovalent capture of competitive PCR products: application to HIV-1 detection. Nucleic Acids Res. 21,3469-3472.
45. Schanke J.T., L.M. Quam and B.G. Van Ness 1994 Flip PCR for DNA sequence motif inversion. Biotechniques 16,414-416.
46. Sieg;omg A., M.Lehmann, C. Platzer, F. Emmrich and H.D.Volk. 1994 A novel multispecific competitor fragment for quantitative PCR analysis of cytokine gene expression in rat. J. Immunol. Methods 177, 23-28.
47. Willard M.F., Stephen J.W., and Kent E.V. Quantitative PT-PCR: pitfalls and potential. 1999 Biotechniques 26,112-125.
