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

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

doi:10.21769/BioProtoc.4810

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

Bio Protoc. 2023 Sep 5;13(17):e4810. doi: 10.21769/BioProtoc.4810.

Authors

Yingying Song^{1

2}, Xinlei Guo², Jian Wu², Jianli Liang², Runmao Lin², Zifu Yan¹, Xiaowu Wang²

Affiliations

¹ College of Horticulture, Henan Agricultural University, Zhengzhou, China.
² Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China.

Abstract

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.

Keywords: Brassica rapa; Guard cells; In situ RT-PCR; Low abundance; Quantitative analysis.