Global cDNA amplification combined with real-time RT-PCR: accurate quantification of multiple human potassium channel genes at the single cell level.
We have developed a sensitive quantitative RT-PCR procedure suitable for the analysis of small samples, including single cells, and have used it to measure levels of potassium channel mRNAs in a panel of human tissues and small numbers of cells grown in culture. The method involves an initial global amplification of cDNA derived from all added polyadenylated mRNA followed by quantitative RT-PCR of individual genes using specific primers. In order to facilitate rapid and accurate processing of samples, we have adapted the approach to allow use of TaqMan real-time quantitative PCR. We demonstrate that the approach represents a major improvement over existing conventional and real-time quantitative PCR approaches, since it can be applied to samples equivalent to a single cell, is able to accurately measure expression levels equivalent to less than 1/100th copy/cell (one specific cDNA molecule present amongst 10(8) total cDNA molecules). Furthermore, since the initial step involves a global amplification of all expressed genes, a permanent cDNA archive is generated from each sample, which can be regenerated indefinitely for further expression analysis.
Al-Taher
A
A
School of Biological Sciences, G.38 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK.
Bashein
A
A
Nolan
T
T
Hollingsworth
M
M
Brady
G
G
D001940:Breast / Q000166:cytology / Q000378:metabolism; D018076:DNA, Complementary; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D010920:Placenta / Q000166:cytology / Q000378:metabolism; D015221:Potassium Channels / Q000235:genetics* / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014599:Uterus / Q000166:cytology / Q000378:metabolism
D018076:DNA, Complementary; D015221:Potassium Channels; D011506:Proteins; D012333:RNA, Messenger