Nucleic Acids Research, 2002, Vol. 30, No. 17 e89
© 2002 Oxford University Press
Improved quantitative real-time RT–PCR for expression profiling of individual cells
University Laboratory of Physiology and MRC Anatomical Neuropharmacology Unit, Department of Pharmacology, Oxford University, Parks Road, Oxford OX1 3PT, UK
*Tel: +44 1865 282490; Fax: +44 1865 272469; Email: birgit.liss{at}physiol.ox.ac.uk
The real-time quantitative polymerase chain reaction (rtqPCR) has overcome the limitations of conventional, time-consuming quantitative PCR strategies and is maturing into a routine tool to quantify gene expression levels, following reverse transcription (RT) of mRNA into complementary DNA (cDNA). Expression profiling with single-cell resolution is highly desirable, in particular for complex tissues like the brain that contain a large variety of different cell types in close proximity. The patch-clamp technique allows selective harvesting of single-cell cytoplasm after recording of cellular activity. However, components of the cDNA reaction, in particular the reverse transcriptase itself, significantly inhibit subsequent rtqPCR amplification. Using undiluted single-cell cDNA reaction mix directly as template for rtqPCR, I observed that the amplification kinetics of rtqPCRs were dramatically altered in a non-systematic fashion. Here, I describe a simple and robust precipitation protocol suitable for purification of single-cell cDNA that completely removes inhibitory RT components without detectable loss of cDNA. This improved single-cell real-time RT–PCR protocol provides a powerful tool to quantify differential gene expression of individual cells and thus could complement global microarray-based expression profiling strategies.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. Grundemann, F. Schlaudraff, O. Haeckel, and B. Liss Elevated {alpha}-synuclein mRNA levels in individual UV-laser-microdissected dopaminergic substantia nigra neurons in idiopathic Parkinson's disease Nucleic Acids Res., April 1, 2008; 36(7): e38 - e38. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Cikos, P. Rehak, S. Czikkova, J. Vesela, and J. Koppel Expression of adrenergic receptors in mouse preimplantation embryos and ovulated oocytes Reproduction, June 1, 2007; 133(6): 1139 - 1147. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Falconi, K. Oizumi, and J. E. Aubin Leukemia Inhibitory Factor Influences the Fate Choice of Mesenchymal Progenitor Cells Stem Cells, February 1, 2007; 25(2): 305 - 312. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. C. Amberg and L. F. Santana Kv2 channels oppose myogenic constriction of rat cerebral arteries Am J Physiol Cell Physiol, August 1, 2006; 291(2): C348 - C356. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Aponte, C.-C. Lien, E. Reisinger, and P. Jonas Hyperpolarization-activated cation channels in fast-spiking interneurons of rat hippocampus J. Physiol., July 1, 2006; 574(1): 229 - 243. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Zhang, M. Chan, and J. E Aubin Pleiotropic effects of the steroid hormone 1,25-dihydroxyvitamin D3 on the recruitment of mesenchymal lineage progenitors in fetal rat calvaria cell populations. J. Mol. Endocrinol., June 1, 2006; 36(3): 425 - 433. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Suslov and D. A. Steindler PCR inhibition by reverse transcriptase leads to an overestimation of amplification efficiency Nucleic Acids Res., November 27, 2005; 33(20): e181 - e181. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. F. Holmes, C. D. Braastad, P. Mitra, C. Hampe, D. Doenecke, W. Albig, J. L. Stein, A. J. van Wijnen, and G. S. Stein Coordinate Control and Selective Expression of the Full Complement of Replication-dependent Histone H4 Genes in Normal and Cancer Cells J. Biol. Chem., November 11, 2005; 280(45): 37400 - 37407. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Devidze, J. A. Mong, A. M. Jasnow, L.-M. Kow, and D. W. Pfaff Sex and estrogenic effects on coexpression of mRNAs in single ventromedial hypothalamic neurons PNAS, October 4, 2005; 102(40): 14446 - 14451. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Miele, C. D. Braastad, W. F. Holmes, P. Mitra, R. Medina, R. Xie, S. K. Zaidi, X. Ye, Y. Wei, J. W. Harper, et al. HiNF-P Directly Links the Cyclin E/CDK2/p220NPAT Pathway to Histone H4 Gene Regulation at the G1/S Phase Cell Cycle Transition Mol. Cell. Biol., July 15, 2005; 25(14): 6140 - 6153. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Wagatsuma, H. Sadamoto, T. Kitahashi, K. Lukowiak, A. Urano, and E. Ito Determination of the exact copy numbers of particular mRNAs in a single cell by quantitative real-time RT-PCR J. Exp. Biol., June 15, 2005; 208(12): 2389 - 2398. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Lu, D. Henderson, L. Liu, P. H. Reinhart, and S. A. Simon TRPV1b, a Functional Human Vanilloid Receptor Splice Variant Mol. Pharmacol., April 1, 2005; 67(4): 1119 - 1127. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Becker, P. K. Hayes, and A. E. Walsby Different gvpC length variants are transcribed within single filaments of the cyanobacterium Planktothrix rubescens Microbiology, January 1, 2005; 151(1): 59 - 67. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Tian, L. Cao, Y. Tan, S. Williams, L. Chen, T. Matray, A. Chenna, S. Moore, V. Hernandez, V. Xiao, et al. Multiplex mRNA assay using electrophoretic tags for high-throughput gene expression analysis Nucleic Acids Res., September 8, 2004; 32(16): e126 - e126. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Kang, V. H. Routh, E. V. Kuzhikandathil, L. D. Gaspers, and B. E. Levin Physiological and Molecular Characteristics of Rat Hypothalamic Ventromedial Nucleus Glucosensing Neurons Diabetes, March 1, 2004; 53(3): 549 - 559. [Abstract] [Full Text] [PDF]
|
|