Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes

Abstract

We describe here a new method for highly efficient detection of microRNAs by northern blot analysis using LNA (locked nucleic acid)-modified oligonucleotides. In order to exploit the improved hybridization properties of LNA with their target RNA molecules, we designed several LNA-modified oligonucleotide probes for detection of different microRNAs in animals and plants. By modifying DNA oligonucleotides with LNAs using a design, in which every third nucleotide position was substituted by LNA, we could use the probes in northern blot analysis employing standard end-labelling techniques and hybridization conditions. The sensitivity in detecting mature microRNAs by northern blots was increased by at least 10-fold compared to DNA probes, while simultaneously being highly specific, as demonstrated by the use of different single and double mismatched LNA probes. Besides being highly efficient as northern probes, the same LNA-modified oligonucleotide probes would also be useful for miRNA in situ hybridization and miRNA expression profiling by LNA oligonucleotide microarrays.

Figure 1

Comparison of LNA2- and LNA3-modified oligonucleotide probes with DNA probes in the detection of the low-abundant miR171 in A.thaliana flowers and leaves by northern blot analysis. Total RNAs (40 μg/sample) from A.thaliana flowers (lane 1) or leaves (lane 2) were electrophoresed on 12% polyacrylamide gel under denaturing conditions, blotted and hybridized with ³²P-labelled miR171LNA2 (A), miR171LNA3 (B) and miR171DNA (C) oligonucleotide probes at 37°C. The membranes were washed at low stringency. The gel loading controls are shown from ethidium bromide staining of the rRNAs (bottom panels). M denotes the RNA molecular weight marker of 20–80 nucleotides.

Figure 2

Assessment of the sensitivity of LNA3-modified probes compared with DNA probes in the detection of miR171 and miR319 in A.thaliana flowers by northern blot analysis. Two duplicate dilution series of A.thaliana total RNA from 100 to 2.5 μg were electrophoresed on 12% polyacrylamide gel under denaturing conditions, blotted and hybridized with ³²P-labelled DNA (A) and LNA3 (B) oligonucleotide probes, respectively, at 34°C. The filters were first hybridized with LNA3 and DNA probes specific for mature mir171, washed with low stringency and exposed as indicated. The filters were stripped, exposed for checking that removal of the probes was complete, and then re-hybridized with LNA3 and DNA probes specific for mature miR319, washed with low stringency and exposed as indicated. The gel loading controls are shown from ethidium bromide staining of the rRNAs (bottom panels).

Figure 3

Improved sensitivity and specificity in the detection of miR171 in A.thaliana flowers and leaves by northern blot analysis using an LNA3-modified oligonucleotide probe. Total RNAs (20 μg/sample) from A.thaliana seedlings (lane 1), leaves (lane 2), flowers (lane 3) and TCV (Turnip crinkle virus)-infected leaves (lane 4) were electrophoresed on 12% polyacrylamide gel under denaturing conditions, blotted and hybridized with ³²P-labelled miR171LNA3 (A), miR171LNA3/2MM (B), miR171DNA (C) and TCVLNA3 oligonucleotide probes at 42°C (D). Stringent washes were carried out in 0.1× SSC, 0.1% SDS at 65°C twice for 5 min. The gel loading controls are shown from ethidium bromide staining of the rRNAs (bottom panels).

Figure 4

Assessment of the specificity of LNA3-modified probes using perfect match and different mismatched probes in the detection of miR171 in A.thaliana flowers and leaves by northern blot analysis. Total RNAs (20 μg/sample) from A.thaliana flowers (lane 1) and leaves (lane 2) were electrophoresed on 12% polyacrylamide gel under denaturing conditions, blotted and hybridized with ³²P-labelled miR171LNA3 (A), miR171LNA3/2MM (B), miR171LNA3/MM11 (C), miR171LNA3/MM8 (D) and miR171LNA3/MM14 (E) LNA-modified oligonucleotide probes at 45°C. The filters were washed at low stringency (upper panels) and high stringency (middle panels). The gel loading controls are shown from ethidium bromide staining of the rRNAs (bottom panels). (F) Validation of the perfect match and the different mismatched LNA probes using complementary DNA oligonucleotide targets. The end-labelled LNA-modified probes were hybridized to their respective perfect match DNA oligonucleotide targets as well as a DNA oligonucleotide corresponding to the mature miRNA171 sequence.

Figure 5

Northern blot analysis of three mouse and two A.thaliana microRNAs using LNA3-modified oligonucleotide probes. (A–D) Total RNAs (20 μg/sample) from mouse brain (lane 1), liver (lane 2) and A.thaliana flowers (lane 3) were electrophoresed on 12% polyacrylamide gels under denaturing conditions, blotted and hybridized with ³²P-labelled miR122aLNA3 (A), miR128LNA3 (B), miR124LNA3 (C) LNA probes as well as TCVLNA3 as a negative control probe (D) at 45°C. (E and F) Total RNAs (20 μg/sample) from A.thaliana flowers (lane 1), leaves (lane 2) and N.benthamiana flowers (lane 3), leaves (lane 4) were electrophoresed on 12% polyacrylamide gel under denaturing conditions, blotted and hybridized with ³²P-labelled miR167LNA3 (E), miR161LNA3 (F) oligonucleotide probes at 45°C. The filters were washed at low stringency. The gel loading controls are shown from ethidium bromide staining of the rRNAs (bottom panels).