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

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 mul 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.

Figure 1

Schematic showing stem-loop RT-PCR miRNA assays. A. Stem-loop RT followed by end-point PCR. Stem-loop RT primers bind to the 3' portion of miRNA molecules, initiating reverse transcription of the miRNA. Then, the RT product is amplified using a miRNA specific forward primer and the universal reverse primer. B. SYBR Green I assay. C. Universal ProbeLibrary (UPL) probe assay. Highlighted in yellow is the UPL probe #21 binding site.

Figure 2

The sensitivity of the stem-loop RT-PCR assay. A. Gel blot analyses of miR156, miR159 and miR167 expression. AS, antisense probe; S, sense probe. B. Stem-loop RT-PCR analyses of miR156, miR159 and miR167 expression. The amounts of RNA used for reverse transcription reactions are indicated on the top. PCR cycle numbers are indicated on the left.

Figure 3

The specificity of the stem-loop RT-PCR assay. A. Gel blot analysis of pri- and mature miR156 expression. High molecular weight (H) and low molecular weight (L) RNA were purified from 20 μg of total RNA, separated by electrophoresis, transferred and hybridised with miR156 antisense probe. B. Stem-loop RT-PCR analyses of miR156 expression. 200 ng total RNA (T), high molecular weight RNA purified from 200 ng total RNA (H) and low molecular weight RNA purified from 200 ng total RNA (L) were subjected to stem-loop RT PCR. The amount of input RNA as measured by NanoDrop is indicated below. PCR cycle numbers are indicated to the left. SA, shoot apex; S, stem: L, leaf.

Figure 4

Detection of miRNAs in tissue and phloem sap. A. Gel blot analyses of miRNA expression in pumpkin shoot apex (SA), leaf (L) and stem (S). An ethidium bromide-stained prominent band of tRNA was used as the loading control (LC). B. Stem-loop end-point RT-PCR analyses of miRNA expression. miRNAs were amplified using 28 cycles of PCR. Pumpkin RUBISCO (CmRBS) mRNA was amplified using 30 cycles of standard PCR. C. Stem-loop end-point RT-PCR analyses of miRNA expression in pumpkin phloem sap. The number of PCR cycles is indicated on the top. miR156, miR159 and miR167, but not miR171 were detected.

Figure 5

miRNA SYBR Green I assay. Real-time PCR amplification profiles of miR156, miR159 and miR167. The amounts of RNA used for stem-loop pulsed RT were as follows: 1, 20 ng; 2, 2 ng; 3, 200 pg; 4, 20 pg; 5, 20 ng minus-RT control; 6, water control.

Figure 6

miRNA UPL probe assay. A. SYBR Green I assay for miR166. Negative control reactions (minus-RT and water) produced detectable amplicons after 40 PCR cycles. B. UPL probe assay for miR166. No fluorescence was detected in the negative control reactions after 45 cycles of PCR. C. UPL probe assay amplification products for miR166 separated by gel electrophoresis on 4% agarose showing specific and non-specific amplification bands obtained after 45 cycles of PCR. Arrowhead indicates the expected size of amplicons. 1, 20 ng RNA; 2, 2 ng RNA; 3, 200 pg RNA; 4, 20 pg RNA; 5, 20 ng RNA minus-RT control; 6, water control.

Figure 7

Expression of miRNAs in vascular tissue and phloem sap. A. Expression of miRNAs in pumpkin vascular tissue (VT) and phloem sap (PS) detected by miRNA UPL assay. Samples containing 10 ng total RNA isolated from pumpkin vascular bundles and phloem sap each were subjected to stem-loop RT reactions and subsequent UPL qPCR. The PCR was performed in three replicates and miRNA expression was normalized against CmPP16 and expressed as a ratio with vascular tissue miR159 expression, which was set arbitrarily at 1. B. Expression of miRNAs detected by gel blot analyses.