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ERIC Number: EJ1106295
Record Type: Journal
Publication Date: 2016
Pages: 7
Abstractor: As Provided
ISSN: ISSN-1470-8175
RT-qPCR Demonstrates Light-Dependent AtRBCS1A and AtRBCS3B mRNA Expressions in "Arabidopsis thaliana" Leaves
Chang, Ming-Mei; Li, Anna; Feissner, Robert; Ahmad, Talal
Biochemistry and Molecular Biology Education, v44 n4 p405-411 Jul-Aug 2016
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 is 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[superscript -??C][subscript T] method and the collected "C"[subscript T]s. In the third week, class "C"[subscript T]s, melting curves, students' calculations, and factors affecting the reliability of RT-qPCR results were summarized and discussed. Students' results show that (1) relatively pure and intact RNA samples are obtained; (2) ACTIN2 is a better reference gene than the 18S rRNA; and (3) 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.
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Publication Type: Journal Articles; Reports - Research
Education Level: N/A
Audience: N/A
Language: English
Sponsor: N/A
Authoring Institution: N/A
Grant or Contract Numbers: N/A