Measurement of gene expression from single adherent cells and spheroids collected using fast electrical lysis.
The cytosol of a single adherent cell was collected by the electrical cell lysis method with a Pt-ring capillary probe, and the cellular messenger RNA (mRNA) was analyzed at a single-cell level. The ring electrode probe was positioned 20 microm above the cultured cells that formed a monolayer on an indium-tin oxide (ITO) electrode, and an electric pulse with a magnitude of 40 V was applied for 10 micros between the probe and the ITO electrodes in an isotonic sucrose solution. Immediately after the electric pulse, less than 1 microL of the lysed solution was collected using a micro-injector followed by RNA purification and first strand cDNA synthesis. Real-time PCR was performed to quantify the copy numbers of mRNA encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression inside the single cell. The average copy numbers of GAPDH mRNA collected by the electrical cell lysis method were found to be comparable to those obtained by a simple capillary suction method. Although single-cell analysis has already been demonstrated, we have shown for the first time that the fast electrical cell lysis can be used for quantitative mRNA analysis at the single-cell level. This electrical cell lysis method was further applied for the analysis of mRNA obtained from single spheroids-the aggregated cellular masses formed during the three-dimensional culture -- as a model system to isolate small cellular clusters from tissues and organs.
Graduate School of Environmental Studies, Tohoku University, Sendai, Japan.
Yuji
Y
Nashimoto
Yasufumi
Y
Takahashi
Takeshi
T
Yamakawa
Yu-Suke
YS
Torisawa
Tomoyuki
T
Yasukawa
Takahiro
T
Ito-Sasaki
Masaki
M
Yokoo
Hiroyuki
H
Abe
Hitoshi
H
Shiku
Hideki
H
Kambara
Tomokazu
T
Matsue
D002448:Cell Adhesion; D002457:Cell Extracts; D002460:Cell Line; D016764:Cell Polarity; D004583:Electrons; D018628:Gene Dosage; D015870:Gene Expression; D006801:Humans; D039641:Integrin beta Chains / Q000235:genetics; D016133:Polymerase Chain Reaction; D013997:Time Factors; D014158:Transcription, Genetic / Q000235:genetics
D002457:Cell Extracts; D039641:Integrin beta Chains