Title: Application of reverse transcription-PCR and real-time PCR in nanotoxicity research.

Authors: Mo, Yiqun; Wan, Rong; Zhang, Qunwei

Published In Methods Mol Biol, (2012)

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.

PubMed ID: 22975959

MeSH Terms: Animals; Humans; Interleukin-1beta/genetics; Interleukin-1beta/metabolism; Matrix Metalloproteinase 2/genetics; Matrix Metalloproteinase 2/metabolism; Monocytes/drug effects; Monocytes/enzymology; Nanoparticles/toxicity*; Nanotechnology/methods*; RNA, Messenger/genetics; RNA, Messenger/metabolism; RNA/isolation & purification; Rabbits; Real-Time Polymerase Chain Reaction/methods*; Reverse Transcriptase Polymerase Chain Reaction/methods*; U937 Cells