利用原位RT-PCR技术检测油菜保护细胞特异性基因表达的优化方案

Yingying Song, Xinlei Guo, Jian Wu, Jianli Liang, Runmao Lin, Zifu Yan, Xiaowu Wang
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摘要

由于白菜(Brassica rapa)的遗传转化尚未很好地发展,原位RT-PCR是检测保护细胞特异性基因的有价值的选择。本文报道了一种利用油菜FAMA同源基因Bra001929进行原位RT-PCR的优化方案。FAMA在拟南芥中已被证实在保卫细胞中特别表达。我们设计了特异性RT-PCR引物,并从(a)逆转录时间、(b)阻断时间、(c)抗原抗体孵育时间、(d)洗涤温度等方面对方案进行了优化。我们的方法提供了一种灵敏有效的原位RT-PCR方法,可以通过在油菜保护细胞中提高低丰度转录本的水平来检测细胞中的低丰度转录本。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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An Optimized Protocol for Detecting Guard Cell-specific Gene Expression by in situ RT-PCR in Brassica rapa.

Since the genetic transformation of Chinese cabbage (Brassica rapa) has not been well developed, in situ RT-PCR is a valuable option for detecting guard cell-specific genes. We reported an optimized protocol of in situ RT-PCR by using a FAMA homologous gene Bra001929 in Brassica rapa. FAMA in Arabidopsis has been verified to be especially expressed in guard cells. We designed specific RT-PCR primers and optimized the protocol in terms of the (a) reverse transcription time, (b) blocking time, (c) antigen-antibody incubation time, and (d) washing temperature. Our approach provides a sensitive and effective in situ RT-PCR method that can detect low-abundance transcripts in cells by elevating their levels by RT-PCR in the guard cells in Brassica rapa.

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