Abstract
Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), a major development in PCR technology, is a powerful and sensitive gene analysis technique that has revolutionized the field of gene expression assays. In this chapter, we describe in detail RNA extraction, reverse transcription (RT), and relative quantification of genes forming the endocannabinoid system in different experimental models. In particular, we here provide specific and sensitive assays to be used to assess gene expression of the endocannabinoid system components in mouse, rat, or human samples.
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References
Mullis KB, Faloona FA (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed Chain reaction. Methods Enzymol 155:335–350
Orlando C, Pinzani P, Pazzagli M (1998) Developments in quantitative PCR. Clin Chem Lab Med 36:255–269
Lockey C, Otto E, Long Z (1998) Real-time fluorescence detection of a single DNA molecule. Biotechniques 24:744–746
Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193
Pfaffl MW, Hageleit M (2001) Validities of mRNA quantification using recombinant RNA and recombinant DNA external calibration curves in real-time RT-PCR. Biotechnol Lett 23:275–282
Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159
Pucci M, Micioni Di Bonaventura MV, Zaplatic E, Bellia F, Maccarrone M, Cifani C, D’Addario C (2019) Transcriptional regulation of the endocannabinoid system in a rat model of binge-eating behavior reveals a selective modulation of the hypothalamic fatty acid amide hydrolase gene. Int J Eat Disord 52: 51–60
Pucci M, Pasquariello N, Battista N et al (2012) Endocannabinoids stimulate human melanogenesis via type-1 cannabinoid receptor. J Biol Chem 287:15466–15478
Catanzaro G, Battista N, Rossi G et al (2011) Effect of capacitation on the endocannabinoid system of mouse sperm. Mol Cell Endocrinol 343:88–92
Di Francesco A, Falconi A, Di Germanio C et al (2015) Extravirgin olive oil up-regulates CB1 tumor suppressor gene in human colon cancer cells and in rat colon via epigenetic mechanisms. J Nutr Biochem 26:250–258
Ishii K, Fukui M (2001) Optimization of annealing temperature to reduce bias caused by a primer mismatch in multitemplate PCR. Appl Environ Microbiol 67:3753–3755
Radonić A, Thulke S, Mackay IM et al (2004) Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun 313:856–862
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Pucci, M., D’Addario, C. (2023). Assessing Gene Expression of the Endocannabinoid System Components by Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction. In: Maccarrone, M. (eds) Endocannabinoid Signaling. Methods in Molecular Biology, vol 2576. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2728-0_29
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DOI: https://doi.org/10.1007/978-1-0716-2728-0_29
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Publisher Name: Humana, New York, NY
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