Tips From the Bench:
High Throughput miRNA Isolation
The MagMAX™-96 for Microarrays Total RNA Isolation Kit combines TRI Reagent® Solution and the Ambion® MagMAX bead-based technology to recover high-quality RNA ideal for use on microarrays. Now, with a minor protocol modification, miRNAs can be isolated using this kit as well—the time required and the high throughput format remain unchanged.
• Purify high yields of quality RNA including miRNAs from frozen tissues, tissues stored in RNAlater® Solution, and cells
• Recover RNA samples in 20–50 µL
• Suitable for real-time RT-PCR or array analysis
How to Recover Total RNA Containing Small RNAs
The modified MagMAX for Microarrays protocol was used with mouse liver stored in RNAlater Solution, frozen mouse heart, and frozen mouse spleen. Briefly, tissues were homogenized in TRI Reagent Solution at a concentration of 50 mg/mL. Bromochloropropane was then added to remove proteins, lipids, and DNA. One-fifth of the aqueous phase was isolated per well using the MagMAX for Microarrays Kit. To recover miRNAs, 1.25 volumes of isopropanol was added to the aqueous phase instead of the standard half volume. The remainder of the protocol was not altered from the original MagMAX for Microarrays protocol.
Analyses of miRNA Content
After RNA isolation, the samples were analyzed on an Agilent® 2100 bioanalyzer and NanoDrop® ND-1000 spectrophotometer (Figure 1). A strong peak representing ~85 nucleotides is observed in all three samples. This peak indicates that small RNAs such as tRNA, 5S rRNA, and miRNAs have been retained in the preparation. Overall, the yields and the RIN suggest good recovery of quality RNA for both the liver and heart samples. The spleen sample appears to contain more degradation products than the heart and liver. This is possibly due to the nature of the tissue and not the isolation procedure, as similar results with spleen were obtained using other isolation methods.
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| Figure 1. Obtain High Quality RNA Using the MagMAX™-96 for Microarrays Total RNA Isolation Kit. Electropherograms were generated with the Agilent® 2100 bioanalyzer. The strong peak at 85 nucleotides likely represents small RNAs such 5S rRNA and tRNA. The peaks at 1800 nucleotides and 3600 nucleotides represent 18S rRNA and 28S rRNA, respectively. The A260/A280 absorbance data was obtained using a NanoDrop® spectrophotometer. |
The presence of miRNAs was further verified using Applied Biosystems TaqMan® MicroRNA Assays (Figure 2). Recovery of miRNAs using the modified MagMAX for Microarrays protocol was compared to traditional mirVana™ miRNA isolation methods using assays for 3 mature miRNAs. As no significant differences were detected between these methodologies, we conclude that this protocol modification supports efficient miRNA isolation for high throughput experiments.
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| Figure 2. TaqMan® MicroRNA Assays Show Comparable Results Between High Throughput Method (MagMAX™ for Microarrays Kit) and Single-Tube Method (mirVana™ miRNA Isolation Kit). Total RNA including miRNA was recovered using either the MagMAX-96 for Microarrays or mirVana miRNA Isolation Kits from the same three mouse tissues. Three miRNA targets were detected in both sample sets using TaqMan MicroRNA Assays. Each RNA sample (10 ng) was assessed by 2-step, real-time RT-PCR as recommended. CT values corresponding to the three test tissues were similar for both isolation methods. This indicates that for a given amount of tissue, the recovery of miRNAs is comparable for the two isolation methods. |
High yields of pure, intact total RNA containing miRNAs can be used in standard real-time RT-PCR assays (TaqMan® Gene Expression Assays) and real-time RT-PCR assays targeting mature microRNAs (TaqMan MicroRNA Assays). The RNA samples can also be used for analysis on mirVana miRNA Bioarrays.
Scientific Contributors
Nathan Harris, Adam Toguchi • Applied Biosystems, Austin, TX
For Research Use Only. Not for use in diagnostic procedures. |
