A Single-Cell-Type Real-Time PCR Method Based on a Modified Patch-Pipette Cell Harvesting System.
Real-time PCR is a powerful tool for quantifying nucleic acid expression. Real-time PCR is conventionally performed at the tissue level to guarantee an abundance of nucleic acid for detection. The precision and reliability of this method, however, is limited by usually being composed of a mixture of different cell types. Single-cell PCR, in contrast, eliminates the purity problem of the cell source. However, use of this method is usually impeded by difficulties in cell harvesting and stringent requirements for processing of very small quantities of nucleic acids. In this study, we combined the advantages of the high purity of selected cells in single-cell PCR with the greater nucleic acid quantities and thus greater ease of tissue-level PCR. The key aspect of our method is to use a modified patch-clamp pipette to harvest several selected cells of the same type. This method is therefore especially useful for cells that can be morphologically or histologically identified such as primary sensory neurons, striated muscle fibers and cells labeled with fluorescent makers.
Department of Physiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Rd., Wuhan, China.|Institute of Brain Research, Huazhong University of Science and Technology, Wuhan, China.
Yuanlong
Y
Song
Department of Physiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Rd., Wuhan, China.
Miaomiao
M
Zhang
Department of Physiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Rd., Wuhan, China.
Xiaoqing
X
Tao
Department of Physiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Rd., Wuhan, China.
Zifen
Z
Xu
Department of Physiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Rd., Wuhan, China.
Liangpin
L
Zhang
Department of Physiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Rd., Wuhan, China.
Yunjie
Y
Zheng
Department of Physiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Rd., Wuhan, China.
Minjie
M
Zhu
Department of Physiology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Rd., Wuhan, China. linlin.gao@foxmail.com.|Institute of Brain Research, Huazhong University of Science and Technology, Wuhan, China. linlin.gao@foxmail.com.
Linlin
L
Gao
D000818:Animals; D002469:Cell Separation / Q000295:instrumentation*; D005260:Female; D009474:Neurons / Q000166:cytology; D018408:Patch-Clamp Techniques; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods*
Cell harvesting; Methods; PCR; Patch-clamp; Single-cell