RT-qPCR demonstrates light-dependent AtRBCS1A and AtRBCS3B mRNA expressions in Arabidopsis thaliana leaves
详细信息 查看全文
- 作者：Ming-Mei Chang ; Anna Li ; Robert Feissner and Talal Ahmad
- 刊名：Biochemistry and Molecular Biology Education
- 出版年：2016
- 出版时间：July/August 2016
- 年：2016
- 卷：44
- 期：4
- 页码：405-411
- 全文大小：453K
- ISSN：1539-3429
文摘
Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is widely used in diagnosis and research to determine specific mRNA expressions in cells. As RT-qPCR applications increase, it's necessary to provide undergraduates hands-on experience of this modern technique. Here, we report a 3-week laboratory exercise using RT-qPCR to demonstrate the light-dependent expressions of AtRBCS1A and AtRBCS3B genes encoding two Arabidopsis thaliana small subunits of the ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). In the first week, students purified and quantified total RNA from leaves of A. thaliana pretreated in the dark for 96 hr and untreated controls. In the second week, RNA samples were separated by formaldehyde gel electrophoresis and used for RT-qPCR. Students calculated expressions of the two genes in dark treated leaves as percentages of those of the controls by using the 2−ΔΔCT method and the collected CTs. In the third week, class CTs, melting curves, students' calculations, and factors affecting the reliability of RT-qPCR results were summarized and discussed. Students' results show that (i) relatively pure and intact RNA samples are obtained; (ii) ACTIN2 is a better reference gene than the 18S rRNA; (iii) the dark treatment reduces both gene expressions to < 1%; (iv) the reduction in the expression of AtRBCS3B is significantly more than that of the AtRBCS1A. Results from pre- and post-lab tests indicate that besides the theory, this exercise helps students learn the applications and associated techniques of RT-qPCR. Future modifications and new experiments that can be developed based on students' learning outcomes and assessment are also discussed.