RT‐qPCR demonstrates light‐dependent AtRBCS1A and AtRBCS3B mRNA expressions in Arabidopsis thaliana leaves

Abstract

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(-ΔΔC) T method and the collected CT s. In the third week, class CT s, 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. © 2016 by The International Union of Biochemistry and Molecular Biology, 44(4):405-411, 2016.

Alternate expression of CONSTANS-LIKE 4 in short days and CONSTANS in long days facilitates day-neutral response in Rosa chinensis

“…Every experiment was conducted with three replicates each with three technical repeats. Semi-quantitative RT-PCR was performed to identify gene expression in Arabidopsis by using the primers listed in Supplementary Table S1 , and Actin2 was used as reference gene ( Chang et al , 2016 ).…”

Identifying the Genes Regulated by AtWRKY6 Using Comparative Transcript and Proteomic Analysis under Phosphorus Deficiency

“…Six ribulose bisphosphate carboxylase proteins (OL40, 41, 42, 43, 44, 45) were downregulated; this is possibly related to high sucrose concentration in 35S:WRKY6-9 shoots, as many genes encoding photosystesubunits and small subunits of Rubisco showed decreased expression when there were high sucrose concentrations in Arabidopsis leaves, resulting in a reduction of photosynthesis [36,37]. Physiological analysis showed that phosphorus deficiency reduces the carboxylase activity of RuBisCO 65.02% [38]. AtRBCS1A exhibits light-dependent expression [39].…”

“…AtRBCS1A exhibits light-dependent expression [39]. Decreased RBCS3B expression causes severe photoinhibition, reflecting the accumulation of ROS, which accelerates the photodamage of PSII and inhibits the repair of PSII in RBCS3B-7 (cosuppression material) [38]. Phosphorus starvation reduces the rate of RuBP regeneration and inhibits the Calvin cycle.…”

Virus induced PhFTRv gene silencing results in yellow-green leaves and reduced cold tolerance in petunia

“…FN014209) was used as internal reference gene to quantify the cDNA abundances. Melting curve analysis is referred to Chang et al (2016), data analysis is carried out by the 2 -ΔΔCT method (Livak and Schmittgen 2001). The sequences of all primers used for qPCR analysis are in Table 1 Suppl.…”