Abstract
Reverse transcription-polymerase chain reaction (RT-PCR) is a relatively simple and inexpensive technique to determine the expression level of target genes and is widely used in biomedical science research including nanotoxicology studies for semiquantitative analysis. Real-time PCR allows for the detection of PCR amplification in the exponential growth phase of the reaction and is much more quantitative than traditional RT-PCR. Although a number of kits and reagents for RT-PCR and real-time PCR are commercially available, the basic principles are the same. Here, we describe the procedures for total RNA isolation by using TRI Reagent, for reverse transcription (RT) by M-MLV reverse transcriptase, and for PCR by GoTaq® DNA Polymerase. And real-time PCR will be performed on an iQ5 multicolor real-time PCR detection system by using iQ™ SYBR Green Supermix.
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References
Product information, Technical Bulletin MB-205. TRI Reagent™ (Sigma, St. Louis, MO), Cat. #T9424
Usage information. M-MLV reverse transcriptase (Promega, Madison, WI), Cat. #M1701
Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Acknowledgments
This work was partly supported by American Lung Association (RG-872-N), American Heart Association (086576D), KSEF-1686-RED-11, Health Effects Institute (4751-RFA-05-2/06-12), CTSPGP 20018 from University of Louisville, T32-ES011564, and ES01443.
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© 2012 Springer Science+Business Media, LLC
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Mo, Y., Wan, R., Zhang, Q. (2012). Application of Reverse Transcription-PCR and Real-Time PCR in Nanotoxicity Research. In: Reineke, J. (eds) Nanotoxicity. Methods in Molecular Biology, vol 926. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-002-1_7
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DOI: https://doi.org/10.1007/978-1-62703-002-1_7
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-001-4
Online ISBN: 978-1-62703-002-1
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