在研究單一細胞分子的特性上,利用單一細胞層級反轉錄多鏈鎖反應是一個非常有效力的技術。利用此技術,在同一顆細胞中可以了解很多基因的表現,如此可以更了解單一細胞的特性。對於一些特異性非常高的細胞,如:背根神經節神經細胞,因為它們具有很複雜的功能及特性,因此要去區分它們的細胞種類是非常困難的。所以若要了解它們的特性及種類,需要更多的分子訊息,而單一細胞層級反轉錄多鏈鎖反應就為一個好的技術。可是此技術仍有很多需要改進的空間,在這個研究中,我嘗試去改善此技術包含在收集細胞的方法、多鏈鎖反應的敏感度及降低操作過程中可能造成的汙染,進而去提高此技術的效力,可以在一個細胞中偵測超過100個基因的表現。藉由改善此技術,我試圖去研究一群表現第三型酸離子通道 (ASIC3) 肌肉訊息傳入神經元的分子特性。結果發現,與其他不表現ASIC3的肌肉訊息傳入神經元比較,有表現ASIC3的肌肉訊息傳入神經元具有非常不同的分子特性。在此研究中,我成功地建立一個適當且有效力的技術去探討一群具有高特異性的神經細胞的分子特性。
Single cell RT-PCR is a powerful tool to explore the molecular identity of single cells. It can determine the expression of many genes in a single cell simultaneously and help to characterize the diverse cell population like dorsal root ganglion DRG neurons. However, there are still some limitations of single cell RT-PCR to obtain a large amount of informative data in gene expression from a single cell. Therefore, I established a new protocol to perform single cell RT-PCR with modification on cell harvesting approach, increased sensitivity of gene determination and reduced contamination. Ideally, this method will be able to detect over 100 genes in a single neuron. I applied this modified single cell RT-PCR to investigate the molecular identity of muscle afferent neurons and showed that ASIC3 expressing muscle afferent neurons had distinct molecular identity from other neurons. The single cell RT-PCR data were consistent with previous studies using immunostaining. In the thesis, I successfully established a powerful technique to investigate the molecular identity of a diverse neuronal population.