Global miRNA Profiling: Higher Sensitivity, Less Sample

TaqMan® Low Density Array Human MicroRNA Panel

  • Streamlined workflow—3 hrs instead of 2 days compared with microarrays
  • Greater sensitivity—3–10 fold less sample (as little as 80 ng) required than with microarrays
  • Wide dynamic range—achieve 6–7 logs dynamic range compared to 3 logs for microarrays
  • High specificity—discriminate between closely related family members differing by as little as a single base
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A Superior Profiling Alternative to Microarray Analysis

The need to measure the expression of hundreds of miRNAs within a given sample led to the use of microarrays for miRNA profiling. However, microarray analysis technology is time consuming, requires large sample inputs, and the resulting data requires specialized analysis. Real-time PCR, using the TaqMan® Low Density Array Human MicroRNA Panel v1.0, can now profile hundreds of miRNAs simultaneously using the highly optimized TaqMan chemistry. In addition, it provides several orders of magnitude greater sensitivity and a significantly broader dynamic range than microarray profiling. The streamlined protocol takes only 3 hours to complete, compared to the 2 day workflow of array analysis.

Profile Hundreds of miRNAs Simultaneously

Each TaqMan Low Density Array Human MicroRNA Panel v1.0 card contains 365 preloaded human miRNA targets, all cataloged in the miRNA miRBase database. An additional 2 controls representing small nucleolar RNAs (snoRNAs), RNU48 (SNORD48) and RNU44 (SNORD44), are preloaded into wells fed by each of the 8 fill ports on each assay card. Used in conjunction with Multiplex RT pools for TaqMan MicroRNA Assays—eight predefined RT primer pools each composed of up to 48 RT primers—the expression of 365 human miRNAs can be quantified simultaneously.

Profiling Has Never Been This Easy or Fast!

Running the preloaded TaqMan Array is a simple, two-step process. During the first step, total RNA (nonenriched, containing small RNA species; e.g., isolated with the mirVana™ miRNA Isolation Kit) is reverse transcribed using predefined RT primers that are specific for only the mature miRNA species. This step has been reduced to eight independent RT reactions using the Multiplex RT pools (see MicroRNA Profiling: Save Time and Sample Using Real-Time PCR), resulting in significant time and sample savings. For this step, microarrays require 1000 ng total RNA, and individual assays require 366 ng total RNA. The Multiplex RT pools allow you to use as little as 80 ng or 10 ng/RT reaction. In the second step, each of the resulting eight RT pools containing cDNA template are diluted, mixed together with TaqMan Universal PCR Master Mix, and loaded into each of the eight fill ports on the TaqMan Array (Figure 1). The card is briefly centrifuged to distribute samples to multiple wells on the array, and then sealed. Within minutes the real-time PCR can be run using the Applied Biosystems 7900HT Fast Real-Time PCR platform. Standard 7900RQ software is used for data analysis.


Figure 1. Fast, Sensitive miRNA Profiling by Real-Time RT-PCR.

Conclusion

The TaqMan Low Density Array Human MicroRNA Panel v1.0 provides a fast, sensitive alternative to microarrays for miRNA profiling. Fully tested with the Applied Biosystems Multiplex RT pools and the Ambion mirVana miRNA Isolation Kit to complete the experimental workflow, miRNA profiling has never been easier.

Note that many of the human targets represented can also be assessed using mouse samples.
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