Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs

Erika Varkonyi-Gasic,Marion Wood,Eric F Walton,Roger P Hellens,Rongmei Wu

doi:10.1186/1746-4811-3-12

Abstract

MicroRNAs (miRNAs) are a class of small non-coding RNAs with a critical role in development and environmental responses. Efficient and reliable detection of miRNAs is an essential step towards understanding their roles in specific cells and tissues. However, gel-based assays currently used to detect miRNAs are very limited in terms of throughput, sensitivity and specificity. Here we provide protocols for detection and quantification of miRNAs by RT-PCR. We describe an end-point and real-time looped RT-PCR procedure and demonstrate detection of miRNAs from as little as 20 pg of plant tissue total RNA and from total RNA isolated from as little as 0.1 μl of phloem sap. In addition, we have developed an alternative real-time PCR assay that can further improve specificity when detecting low abundant miRNAs. Using this assay, we have demonstrated that miRNAs are differentially expressed in the phloem sap and the surrounding vascular tissue. This method enables fast, sensitive and specific miRNA expression profiling and is suitable for facilitation of high-throughput detection and quantification of miRNA expression.

Highlights

MicroRNAs are families of short non-coding transcripts, arising from larger precursors with a characteristic hairpin secondary structure [reviewed in [1]]
A number of specific quantitative reverse transcription-polymerase chain reaction (RT-PCR) techniques were developed and optimised for miRNA detection, including real-time methods based upon reverse transcription (RT) reaction with a stem-loop primer followed by a TaqMan PCR analysis [31,32]
These results suggest that the stem-loop RT-PCR assay is highly specific for mature miRNAs

Summary

Introduction

MicroRNAs (miRNAs) are families of short non-coding transcripts, arising from larger precursors with a characteristic hairpin secondary structure [reviewed in [1]]. MiRNA represent a relatively abundant class of transcripts, their expression levels vary greatly among different cells and tissues Conventional technologies such as cloning, northern hybridization and microarray analysis are widely used but may not be sensitive enough to detect less abundant miRNAs. intensive small RNA sequencing revealed a very complex small RNA population in plants. A number of specific quantitative RT-PCR (qRT-PCR) techniques were developed and optimised for miRNA detection, including real-time methods based upon reverse transcription (RT) reaction with a stem-loop primer followed by a TaqMan PCR analysis [31,32]. Equipment Standard laboratory equipment including a thermal cycler is required for pulsed reverse transcription and end-point PCRs. A real-time thermal cycler is required for SYBR Green I and UPL probe assays.

Perform real-time PCR:

Conclusion

Bartel DP

21. Chen X