Rapid and accurate detection of plant miRNAs by liquid northern hybridization

Abstract

Northern blot analysis is a powerful research tool for discovery, validation and expression of genes, and is currently widely used to detect microRNA (miRNA) accumulation. However, the traditional Northern blot procedure, which is based on a support membrane, is overly elaborate and time-consuming, although it is unsurpassed in accuracy for determining the sizes and amounts of multiple small RNAs sharing high sequence identity. Here we present an alternative method derived from plant miRNAs, liquid Northern hybridization, using fluorescently labeled oligonucleotide probes and characterized by simple and specific miRNA determination and quantitation. The entire detection process is completed within a few hours, and multiple miRNAs can be simultaneously detected in a single experiment.

Keywords: Oryza sativa; liquid Northern hybridization; miRNA; oligonucleotide probes; small RNA.

Figure 1

Non-denaturing polyacrylamide gel electrophoresis of liquid Northern hybridization of Osa-miD156* probes with Osa-miD156 (negative image). Hybridization of Osa-miD156* probe with Osa-miR156 as molecular marker. (a) Hybridization of 10 pmol of Osa-miD156 with different amounts of Osa-miD156* probes. (b) Hybridization of 4 pmol/L of Osa-miD156* probes and 4 pmol/L of Osa-miD156 with different mismatched bases at 42 ºC, 57 ºC and 60 ºC. (c) Effects of different temperatures on hybridization of 4 pmol/L Osa-miD156* probes with 5 pmol/L of Osa-miD156. (d) The effect of hybridization times on hybridization between 4 pmol/L Osa-miD156* probes and 4 pmol/L Osa-miD156.

Figure 2

Liquid hybridization detection of miRNAs from different plant materials. Hybridization of Osa-miD156* probe with Osa-miR156 as molecular marker. (a) Detection of miR156 from rice seedlings in different hybridization buffers. (b) Detection of miR156 from several plant species in buffer 3. (c) Detection of miR156 from different rice tissues. (d) Detection of different miRNAs from rice seedlings. (e) Quantitative analysis of miR156 in rice seedlings by liquid Northern hybridization. Upper: image of miR156 from rice seedlings; lanes 1–8 represent 10 (Std1), 5 (Std2), 2.5 (Std3), 1.25 (Std4), 0.625 (Std5), 0.313 (Std6), 0.156 (Std7) and 0.078 (Std8) pmol, respectively, for creating the standard curve; Lane 9 is a small RNA sample we got from rice seedlings and used 1 ug miRNA (89.69 ng/μL). Lower: image is a quantification using a Bio-Rad gel imaging system.