Abstract
Steady-state mRNA levels are determined by both the rates of transcription and degradation. Regulation of mRNA stability and/or degradation are key factors that can significantly affect mRNA levels and its biological functions. mRNA stability can be measured indirectly after transcription inhibition. This protocol described a rapid and sensitive method of mRNA stability measurement through quantitative reverse transcription PCR (RT-qPCR) after inhibition of RNA transcription by cordycepin in Arabidopsis seedlings.
Keywords: RNA Stability, RNA Degradation, Arabidopsis, RT-qPCR, Cordycepin, Primary MicroRNA Transcripts
Background
The regulation of mRNA stability is a key control point in gene expression regulation. mRNA stability has a profound impact on gene expression, molecular and cellular phenotype, and ultimately, plant development, defense and other biological processes. Multiple methods, e.g., Northern blot analysis, in situ hybridization, can be used to measure mRNA stability after transcription inhibition. In this protocol, we describe a rapid and sensitive method to measure mRNA stability through RT-qPCR after inhibition of transcription by cordycepin. Cordycepin or 3'-deoxyadenosine is an adenosine analogue (see Reference 2). 3'-deoxyadenosine can be incorporated into RNA and inhibits transcription elongation and RNA synthesis due to the absence of a hydroxyl moiety at the 3' position (see Reference 6). We have successfully used this convenient and sensitive method to measure the stability of several low-abundant mRNAs, including primary microRNA transcripts, in Arabidopsis seedlings (Jia et al., 2017). Here we present this protocol with detailed experimental procedures and data analysis methods.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Acknowledgments
We thank Dr. Xuemei Chen and UC Riverside for supporting this work. This work was funded by the National Institutes of Health (GM061146) and Gordon and Betty Moore Foundation (GBMF3046) to Dr. Xuemei Chen. This protocol was adapted from previous works by Lidder et al. (2005), Zhang et al. (2014), and Jia et al. (2017).
Competing interests
The authors declare that there is no conflicts of interest or competing interests.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.