The use of real-time quantitative PCR for the analysis of cytokine mRNA levels

Methods in Molecular Biology

Volumn 820, Issue , 2012, Pages 7-23

  Forlenza, Maria ^a   Kaiser, Thomas ^a   Savelkoul, Huub F J ^a   Wiegertjes, Geert F ^a  

^a WAGENINGEN UNIVERSITY   (Netherlands)

Author keywords

Absolute quantification; Housekeeping gene; Primer efficiency; Real time quantitative PCR; Relative quantification

Indexed keywords

COMPLEMENTARY DNA; CYTOKINE; DNA; FLUORESCENT DYE; MESSENGER RNA; ORGANIC COMPOUND; SYBR GREEN I;

ARTICLE; BIOSYNTHESIS; ESSENTIAL GENE; GENE DOSAGE; GENETICS; ISOLATION AND PURIFICATION; METABOLISM; METHODOLOGY; PLASMID; REAL TIME POLYMERASE CHAIN REACTION; REPRODUCIBILITY; SENSITIVITY AND SPECIFICITY;

CYTOKINES; DNA; DNA, COMPLEMENTARY; FLUORESCENT DYES; GENE DOSAGE; GENES, ESSENTIAL; ORGANIC CHEMICALS; PLASMIDS; REAL-TIME POLYMERASE CHAIN REACTION; REPRODUCIBILITY OF RESULTS; RNA, MESSENGER; SENSITIVITY AND SPECIFICITY;

EID: 84555195899     PISSN: 10643745     EISSN: None     Source Type: Book Series    
DOI: 10.1007/978-1-61779-439-1_2     Document Type: Article

References (15)

1. Nolan, T., Hands, R.E., Bustin, S.A. (2006) Quantifi cation of mRNA using real-time RT-PCR. Nat. Protoc. 1: 1559-1582.
2. Bustin, S.A., Benes, V., Nolan, T., Pfaffl M.W. (2005) Quantitative real-time RT-PCR - a perspective. J. Mol. Endocrinol. 34: 597-601. (Pubitemid 40903586)
3. Pfaffl M.W., Hageleit, M. (2001) Validities of mRNA quantifi cation using recombinant RNA and recombinant DNA external calibration curves in real-time RT-PCR. Biotechnol. Lett. 23: 275-282. (Pubitemid 32162614)
4. Pfaffl M.W. (2001) A new mathematical model for relative quantifi cation in real-time RT-PCR. Nucleic Acids Res. 29: e45.
5. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., Speleman, F. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3: RESEARCH0034.
6. Pfaffl M.W., Tichopad, A., Prgomet, C., Neuvians, T.P. (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper- Excel-based tool using pair-wise correlations. Biotechnol. Lett. 26: 509-515. (Pubitemid 38448421)
7. Latorra, D., Arar, K., Hurley, J.M. (2003) Design considerations and effects of LNA in PCR primers. Mol. Cell. Probes 17: 253-259. (Pubitemid 37346175)
8. Latorra, D., Campbell, K., Wolter, A., Hurley, J.M. (2003) Enhanced allele-specifi c PCR discrimination in SNP genotyping using 3locked nucleic acid (LNA) primers. Hum. Mutat. 22: 79-85. (Pubitemid 36807776)
9. Livak, K.J., Schmittgen, T.D. (2001) Analysis of Relative Gene Expression Data Using Real- Time Quantitative PCR and the 2-[Delta] [Delta]CT Method. Methods 25: 402-408. (Pubitemid 34164012)
10. Pfaffl M.W., Horgan, G.W., Dempfl e, L. (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 30: e36.
11. Liu, W., Saint, D.A. (2002) A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics. Anal. Biochem. 302: 52-59. (Pubitemid 34174608)
12. Ramakers, C., Ruijter, J.M., Deprez, R.H., Moorman, A.F. (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci. Lett. 339: 62-66. (Pubitemid 36263221)
13. Tichopad, A., Dilger, M., Schwarz, G., Pfaffl M.W. (2003) Standardized determination of real-time PCR effi ciency from a single reaction set-up. Nucleic Acids Res. 31: e122.
14. Forlenza, M., de Carvalho Dias, J.D., Vesely, T., Pokorova, D., Savelkoul, H.F., Wiegertjes, G.F. (2008) Transcription of signal-3 cytokines, IL-12 and IFN alpha beta, coincides with the timing of CD8 alpha beta up-regulation during viral infection of common carp (Cyprinus carpio L). Mol. Immunol. 45: 1531-1547. (Pubitemid 351221146)
15. Fleige, S., Pfaffl M.W. (2006) RNA integrity and the effect on the real-time qRT-PCR performance. Mol. Asp. Med. 27: 126-139.