Split single-cell RT-PCR analysis of Purkinje cells.
This protocol details a method for analyzing the expression of multiple genes from a single Purkinje neuron, including the determination of whether the gene expression is monoallelic or biallelic. The protocol describes how to extract a single, living Purkinje cell for reverse transcription, divide the cDNAs into three equal samples and subject those to triplicate amplification of multiple targets by two rounds of PCR (first a multiplex PCR then a gene-specific nested PCR) and finally discriminate the allelic expression of the transcript by direct sequencing of the PCR products. In optimal conditions, this method permits the analysis of the expression of 18 genes in a single Purkinje cell. This protocol can be completed in 5-6 d.
Esumi
Shigeyuki
S
KOKORO-Biology Group, Laboratories for Integrated Biology, Graduate School of Frontier Biosciences, Osaka University 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
Kaneko
Ryosuke
R
Kawamura
Yoshimi
Y
Yagi
Takeshi
T
D000483:Alleles; D000818:Animals; D002469:Cell Separation / Q000379:methods*; D018076:DNA, Complementary; D020869:Gene Expression Profiling / Q000379:methods*; D051379:Mice; D011689:Purkinje Cells / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary