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A simple and accurate method for labeling plant and animal microRNAs
Sponsored,vendor-submitted protocol    Sponsored by Mirus Bio LLC    Published in November 2009 (p.33) DOI: 10.2144/000113306

Abstract

Expression profiling of microRNAs (miRNAs) is a powerful and commonly used methodology for assessing the relative levels of individual miRNAs in specific tissues. To achieve accurate results using miRNA microarray detection strategies, it is important that all sample miRNA species are representatively labeled prior to the hybridization step. The Label IT® miRNA Labeling Kits utilize a chemical alkylation reaction to internally and uniformly Label any miRNA sample, including samples derived from fresh and frozen tissue, and formalin-fixed, paraffin-embedded samples. The following protocol describes the use of Mirus Bio’s Label IT reagents to efficiently Label miRNAs for microarray hybridization. The chemical structure of Label IT® and an outline of the full protocol is shown in Figure 1.

Materials

Label IT miRNA Labeling Kits, version 2 (Mirus Bio LLC, all reagents and buffers used in the following protocol are provided in these kits)

DNase-free and RNase-free water

Microcentrifuge tubes

96−100% ethanol

miRNA-containing sample (purified total or low-molecular weight enriched RNA)

miRNA-specific microarray slides

Slide coverslips

Microarray scanning equipment

Protocol

Label miRNA containing sample

1. Set up the labeling reaction as follows. This reaction can be scaled up or down for different RNA input amounts. Add the reagents in the order listed. Add the Label IT reagent last. If using more than one color/label, set up each labeling reaction separately.

2. Incubate the reaction at 37°C for 1 h.

3. Stop the labeling reaction by adding 0.1 volume of 10× STOP Reagent to the labeling reaction. Vortex gently to mix.

4. If performing a dual color hybridization, pool the Cy™3 and Cy5 samples together before proceeding to the column purification steps.

5. Place the reactions on ice or store at ≤-20°C until you are ready to proceed with the purification step.

Purify labeled miRNA using purification columns

Figure 1. Label IT chemical structure and protocol. The Label IT Cy™3, Cy™5, and biotin reagents are composed of three regions: the Cy3, Cy5, or biotin label (green); the linker (yellow), which facilitates electrostatic interactions with nucleic acids; and the reactive alkylating group (blue) that covalently attaches the Label IT reagents to any reactive heteroatom within miRNAs. The covalent attachment of the Label IT reagents to miRNA does not alter the structure or downstream hybridization performance of the miRNA, and can be used for microarray hybridization.

1. Add 0.1 volume of Column Binding Buffer to the labeled sample.

2. Add 2.5 volumes of ethanol (96−100%) to the labeled sample. Mix well.

3. Apply the labeled sample to the provided Purification Column (with collection tube).

Do not apply more than 720 L to the column for any centrifugation. If the sample volume is greater than 720 L, repeat centrifugation until all of the labeled sample has passed through the column.

4. Centrifuge for 30 s at 11,000× g.

5. Discard the flow-through from the collection tube. Use the same collection tube for all wash steps.

6. Apply 600 L of Column Wash Buffer to the column.

7. Centrifuge the column for 30 s at 11, 000× g. Discard the flow-though in the collection tube.

8. Apply 200 L of Column Wash Buffer to the column.

9. Centrifuge for 2 min at 11,000× g. Discard the flow-though in the collection tube.

10. Transfer the column to the 1.5 ml elution tube.

11. Apply 28.5 L Elution Buffer to the column (when using a 22 × 60 mm standard LifterSlip™).

12. Centrifuge for 1 min at 11, 000× g.

13. Remove the column from the elution tube. The purified labeled sample is now ready to use.

Perform miRNA microarray hybridization

This section includes recommendations and suggestions for miRNA microarray hybridizations. Due to the variability in microarray slides, surface chemistries, capture sequences, and manufacturing processes, the hybridization process should be optimized specifically for the microarray being used. Ensure that the microarray contains verified capture sequences that are complementary to directly labeled miRNA species. MicroRNAs labeled using Label IT are versatile and likely to work with many other conditions and buffers.

1. Add 28.5 L 2× Hybridization Solution and mix well before hybridization.

2. Denature the labeled sample in 1× Hybridization Solution at 65°C for 3 min.

3. Hybridize labeled RNA samples according to the following recommendations:

4. Hybridize at ~37°C, for ~16 h (overnight).

Wash the array

1. Wash twice with 1× SSC, 0.1% SDS at 37°C, 2 × 5 min each.

2. Wash once with 1× SSC at 37°C, 2 × 5 min each.

3. Wash with 0.1× SSC at room temperature, 2 × 1 min each.

4. Treat the arrays as follows:



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