Integrated on-capillary instrumentation for gene expression measurement directly from cells.
We studied the fundamental instrumental issues relevant to a capillary-based integrated system to measure expression of a specific gene directly from cells. Samples were introduced into a capillary by use of a syringe pump. All reactions were carried out in a microthermocycler, where a part of the capillary having 1 microL inner volume was used as a reaction vessel. First, cells were lysed by heating to release RNA, followed by deoxyribonuclease (DNase) treatment. Then, reverse transcription-polymerase chain reaction (RT-PCR) was performed to obtain amplified products from the targeted mRNA. Finally, the product was verified by capillary electrophoresis (CE) with laser-induced fluorescence detection. The whole protocol was completed in the system in 3 h. PCR product from beta-actin mRNA in 16 human lymphoblast cells was obtained with a signal-to-noise ratio (S/N) of 3400 +/- 730 (n = 3). Therefore, the system is reproducible and sensitive enough to measure gene expression from a single cell. We show that the amplified fragment from breast cancer-specific mRNA was obtained from cells of breast cancer cell line, but was not obtained from cells of hepatoma cell line. These results therefore lay the foundations for future CE or microchip instrumentation for high-throughput automated gene-expression analysis.
Matsunaga
Hiroko
H
Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, IA 50011, USA.
Anazawa
Takashi
T
Yeung
Edward S
ES
D000199:Actins / Q000235:genetics; D045744:Cell Line, Tumor; D002477:Cells / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D020869:Gene Expression Profiling / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D012333:RNA, Messenger / Q000032:analysis / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000199:Actins; D012333:RNA, Messenger