Detecting miRNA & siRNA
In the past few years, interest in the identification, detection, and use of small RNA molecules has exploded. This interest has primarily stemmed from two interrelated lines of research. In one, small double-stranded RNAs (dsRNAs) called small interfering RNAs (siRNAs) have been used to silence the expression of specific genes at the post transcriptional level by a pathway known as RNA interference (RNAi). In the other, numerous small regulatory RNA molecules, referred to as microRNAs (miRNAs), have been shown to regulate target gene expression in various organisms. It is now becoming apparent that siRNAs and miRNAs are related molecules, sharing common processing pathways and maybe even functional mechanisms.
Currently, most miRNA researchers are analyzing miRNA expression patterns by Northern blot, a technique that is relatively insensitive and labor intensive. A few researchers performing gene silencing experiments also use this technique to analyze siRNA levels after RNAi induction, although the majority of researchers performing gene silencing experiments do not monitor siRNA levels at all. This may be due in large part to the inherent difficulties in detecting small RNAs with standard techniques. Here we discuss an optimized technique for the detection and quantitation of small RNAs. This technique allows easier monitoring of miRNA expression and siRNA levels in a variety of sample types and is considerably more sensitive than Northern analysis.
Solution Hybridization Improves Sensitivity
To examine the sensitivity of the assay, decreasing amounts of a 21 nt RNA, corresponding to the antisense strand of an siRNA known to target GAPDH, were spiked into a yeast RNA sample and then analyzed by the solution hybridization method. In brief, the samples were mixed with a 29 nt high specific activity radiolabeled probe (5 x 104 cpm) and hybridization buffer. After heat denaturation, each mixture was incubated at 42°C to hybridize the probe to the complementary siRNA target strand. Unhybridized RNA species and excess RNA probe were removed by a brief ribonuclease digestion. Protected target RNA fragments were recovered in the same tube using a reagent that simultaneously inactivates the ribonuclease and precipitates the nucleic acid. The protected RNA was then resuspended with gel loading buffer, analyzed on a denaturing polyacrylamide gel, and exposed to film. Figure 1A demonstrates that the assay was able to detect as little as 50 attomoles of the 21 nt target RNA after a two hour exposure.
Figure 1. Sensitive and Specific Detection of MicroRNAs. (A) The indicated amounts of a 21 nt antisense GAPDH siRNA were spiked into 4 µg of yeast RNA and detected with the mirVana™ miRNA Detection Kit using a 29 nt long probe prepared with the mirVana miRNA Construction Kit. Protected RNA fragments (19 nt) were analyzed on a 15% denaturing polyacrylamide gel. (B) The same experiment as in Panel A with 200 attomoles of sense or antisense GAPDH siRNA radiolabeled probes specific for each strand..
Specificity of the Assay
To further investigate assay specificity, miR-16 miRNA was detected in mouse kidney total RNA using three different probes. One probe was perfectly complementary to the target (miR-16), a second included three mismatched nucleotides (mir-16 mut), and a third included four additional A residues between the 22 nt sequence complementary to mir-16 and the leader sequence (mir-16 + 4). The mir-16 and mir-16 + 4 probes were both able to detect miR-16 miRNA in the RNA sample, whereas the signal was completely abolished with mir-16 mut probe (Figure 2). This result demonstrates that the assay has the required specificity for detecting small RNAs in total RNA samples.
Detecting Multiple Small RNAs in the Same Sample
Figure 2. miRNA Expression in Mouse Kidney Total RNA. miR-16 and miR-22 expression was analyzed as in Figure 1 with 1 µg of FirstChoice® Total RNA from mouse kidney and 32 nt long probes generated with the mirVana miRNA Probe Construction Kit. mir-16 mut probe (32 nt) carries 3 mismatch mutations (ACG to CGA) corresponding to nucleotides 9 to 11 of the miR-16 miRNA sequence. The mir-16+4 probe (36 nt) carries 4 additional A residues between the 22 nt sequence specific for miR-16 and the 10 nt leader sequence, producing a 26 nt long protected fragment, which is 4 nt longer than that produced by the mir-16 and mir-22 probes.
Figure 3. Single and Multiple Target Detection. The indicated target RNAs were detected in 500, 250, 100 and 50 ng of total RNA from mouse tissues (3.5 hours exposure) or HeLa cells (6 hours exposure) as described in Figures 1 and 2. The probe specific for GAPDH mRNA (39 nt) produces a 29 nt long protected fragment with the same specific activity as the mir-16 protected fragment. miR-16 was detected with the mir-16 +4 probe.
Analysis of miRNA Expression Patterns Across Various Tissue Types
We used the solution hybridization assay to monitor the differential expression of two different miRNAs across mouse tissues (Figure 4). High levels of mir-16 expression were detected in all five tissues tested, with the highest expression levels evident in lung and thymus. These variations across tissues were confirmed by Northern blot analysis with the same mir-16 probe. Interestingly the mir-22 probe showed a completely different pattern of expression. miR-22 was highly expressed in lung and ovary and was present in spleen, thymus, and testicle at levels that would not have been detectable with standard Northern blotting techniques (data not shown). The relative abundance of miR-16 and miR-22 miRNA in mouse lung was also confirmed by multi-target detection with the simultaneous use of the mir-16+4 and mir-22 probes in the assay (Figure 3).
Figure 4. miR-16 and miR-22 Expression in Mouse Tissues. miR-16 and miR-22 miRNAs (22 nt) were detected in 1 µg of FirstChoice® Total RNA from five different mouse tissues using 32 nt long mir-16 or mir-22 probes generated with the mirVana miRNA Probe Construction Kit. The same differential expression of miR-16 across tissues was observed by Northern blot analysis (2 days exposure) or by hybridization in solution (2 hr exposure). RNAs were analyzed on 15% denaturing polyacrylamide gels. As a loading control, the same RNA samples were resolved on a 1.2% denaturing agarose gel and U1 snRNA expression was analyzed by Northern blot.
Simultaneous Detection of siRNA Expression and Target Gene Knockdown
Figure 5. Analysis of GAPDH siRNA Expression and mRNA Knockdown. (A) HeLa cells were transfected with pSilencer 2.0-U6 engineered to express either an siRNA targeting GAPDH or a negative control siRNA (SCR). Three days after transfection, total RNA was isolated and 1 µg was assessed using the mirVana miRNA Detection Kit. Probes to the antisense strand of the GAPDH siRNA were prepared as described in Figure 1. (B) Same experiment with probes specific for GAPDH mRNA or GAPDH siRNA. Both probes had the same specific activity. As a control, GAPDH mRNA expression was also analyzed by Northern blot.
Advantages of the Solution Hybridization Assay
Ambion makes this solution hybridization assay available as the mirVana miRNA Detection Kit. We also offer the mirVana miRNA Probe Construction Kit, for the easy preparation of short RNA probes for this assay as well as for Northern analysis and in situ hybridization.