miRtect-IT™ miRNA Labeling and Detection Kit

• Fast – capture and label miRNA in just over 2 hours
• Sensitive – detect miRNA in as little as 50 ng or less of total RNA
• Specific – measure mature miRNA without an amplification step
• Quantitative – linear detection range of 0.2 - 20 femtomoles
• Convenient – one labeling reaction for detection of any miRNA of interest
• Flexible – detect a variety of small RNAs e.g. siRNA
• Simple – add-and-incubate assay reaction

The miRtect-IT™ miRNA Labeling and Detection Kit is a novel method for the labeling and detection of mature miRNA and small RNAs from total RNA by splinted-ligation technology^†(1,2,4). The splinted-ligation technology is a nucleic acid hybridization assay that uses a miRNA-specific Bridge Oligonucleotide to form base pairs with the miRNA and a Detection Oligonucleotide. The captured miRNA is subsequently ligated to the Detection Oligonucleotide with T4 DNA Ligase (Figure 1).

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Fig. 1. miRtect-IT™ miRNA Labeling and Detection Kit assay procedure. * Residual radiolabeled 14 nucleotide Detection Oligo will be present due to incomplete removal of the 5' end-32P labeled Detection Oligo.

By joining the miRNA to the radiolabeled Detection Oligonucleotide, the assay directly labels miRNAs and avoids the inherent difficulty of detecting small RNAs of 20-24 nucleotides in Northern blots. Quantitative measurement of miRNAs in total RNA samples can be completed within 6 hours.

Assay Procedure Overview

miRtect-IT™ miRNA Labeling and Detection Kit is a four-step procedure for direct labeling of miRNA from nanogram to microgram quantities of total RNA (Figure 1).

Step 1 Detection Oligo Preparation (40 min)
• 5' End-label Detection Oligo with [-³²P]-ATP and OptiKinase™
• Remove unincorporated nucleotide with Clean-Up Column

Step 2 miRNA Capture (15 min)
• Capture miRNA and the radiolabeled Detection Oligo with Bridge Oligonucleotide

Step 3 miRNA Ligation (75 min)
• Ligate the captured miRNA and the radiolabeled Detection Oligo with Ligate-IT™ Premix
• Remove non-ligated Detection Oligo with Clean-Up Mix

Step 4 Detection (3 hr to overnight)
• Separate ligated miRNA on a 12-15% UREA-polyacrylamide gel
• Visualize by X-ray film or a phosphorimaging system

Specificity and Sensitivity
The miRtect-IT™ assay specificity is shown by measuring endogenous miRNAs from 6 different human miRNAs in 4 different tissues and 1 cell culture (Figure 2).

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Fig. 2. Expression analysis of miRNAs in human tissues (A) and tissue-specific miRNAs (B) from 250 ng total RNA using the miRtect-IT™ miRNA Labeling and Detection Kit. The image was developed after 2 hr exposure.

The observed miRNA expression is in agreement with published studies that have characterized tissue-specific miRNA expression. For example, miR-124a expression is brain-specific and miR-133a-1 expression is muscle-specific⁽³⁾.

Quantitative Results
The miRNA detected by miRtect-IT™ is quantitative and shows correlation to Northern blot data – with a 50-fold better detection sensitivity (Figure 3).

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Fig. 3. Quantitative detection of miR-21 using the miRtect-IT™ miRNA Labeling and Detection Kit (A, upper) and Northern blot (A, lower). Both assays were performed in parallel using HeLa cell total RNA with the indicated amounts. The image was quantified by phosphorimager analysis (B). Reprinted with permission.

Linear Detection
The miRtect-IT™ assay has a linear detection range of 0.2-20 femtomoles based on measurement of synthetic miRNAs (Figure 4). The amount of total RNA required per assay depends on the abundance of the miRNA of interest (Figure 5). The recommended protocol allows up to 8 μl of total RNA per assay reaction. A typical assay reaction uses 0.5 - 4 μg of RNA diluted in TE Buffer or RNase-Free Water.

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Fig. 4. Linear detection range of the miRtect-IT™ miRNA Labeling and Detection Kit. Assay was performed using the indicated amounts of a synthetic oligonucleotide of mature miRNA sequence. The image was developed after 2 hr exposure. Reprinted with permission.

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Fig. 5. Detection sensitivity of the miRtect-IT™ miRNA Labeling and Detection Kit. Assays were performed in parallel using human muscle total RNA and human brain total RNA with the indicated amounts. Total RNAs used for experiments in Figure 2 came from different sources. The image was developed after 2 hr exposure.

Kit Components
Detection Oligo
OptiKinase™
10X OptiKinase™ Reaction Buffer
Clean-Up Columns
10X Capture Buffer
3X Ligate-IT™ Premix
Positive Control
Clean-Up Mix
Gel Loading Dye
RNase-Free Water
Brief Protocol Card
Long Protocol Booklet

The assay requires [-³²P]-ATP (6000 Ci/mmol, 150mCi/ml). Important: A Bridge Oligonucleotide must be obtained separately.

Bridge Oligonucleotide Design
The Bridge Oligonucleotide is a DNA oligonucleotide complementary to both the Detection Oligo and a specific miRNA at its 5' and 3' ends, respectively (Figure 6). Therefore every Bridge Oligonucleotide should have the same 14 nucleotide sequence at the 5' end, which allows one labeling reaction of the Detection Oligo for detection of any miRNA of interest. In general, addition of unligatable-modifications to the ends of the Bridge Oligonucleotide is not always necessary. However, in some cases, it is desirable to block the 3 ’ end or both the 5' and 3 ' ends of the Bridge Oligonucleotide by incorporating modification(s) such as a C3 spacer, amino modifier, inverted dT, or dideoxy-C. This ensures that unwanted side ligation reactions do not take place. USB recommends use of an unmodified Bridge Oligonucleotide as the first option for the assay.

The Bridge Oligonucleotide requires a standard desalted purification after synthesis. Further purification by HPLC or denatured PAGE is usually not necessary. See Protocol Book for more information on design and preparation of the Bridge Oligonucleotide.

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Fig. 6. Example of Bridge Oligonucleotide sequence design.

Flexible
The miRtect-IT™ assay can be used for detection of other regulatory small RNAs⁽⁵⁾ including plant miRNAs known to be 2´-O-methyl modified at their 3´ ends (Fig. 7A), plant-specific endogenous small RNAs (Fig. 7B), testes-specific PIWI-associated small RNAs (piRNAs) (Fig. 7C), and viral-encoded miRNAs (Fig. 7D).

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Fig. 7. Expression analysis of various classes of endogenous regulatory small RNAs using miRtect-IT™. Arabidopsis miRNAs known to have 2'-O-methylation at 3' ends (A), low abundant Arabidopsis miRNAs, trans-acting siRNAs (ta-siRNAs) and small RNAs (B), mouse PIWI-associated small RNAs (piRNAs) (C) and Marek’s Disease Virus (MDV) encoded miRNAs in infected chicken embryo fibroblasts (D) were detected using the indicated amounts of total RNA in A, B and D and 4 μg of total RNA in C. The image was developed after 24-48 hr exposure. Reprinted with permission.

References

1. Moore, M. J. and Query, C. C. (2000) Methods Enzymol. 317, 109-123.
2. Maroney, P. A., Chamnongpol, S., Souret, F. and Nilsen, T. W. (2007), RNA, 13, 930-936. 
3. Lagos-Quintana, M., Rauhut, R., Yalcin, A., Meyer, J., Lendeckel, W. & Tuschl, T. (2002) Curr. Biol 12, 735–739.
4. Maroney, P. A., Chamnongpol, S., Souret, F. and Nilsen, T. W. (2008) Nature Protocols, 13 (1) 279-287.  
5. Lu, C., Jeong, D-H., Kulkarni, K., Pillay, M., Nobuta, K., German, R., Thatcher, S. R., Maher, C., Zhang, L., Ware, D., Liu, B., Cao, X., Meyers, B. C., and Green, P. J. (2008) Proc. Natl. Acad. Sci. USA 105 (2), 4951-4956.

^†USB Patent Pending

Storage:
Shipped on dry ice. Store at -20°C.