NEW Multiplex RT & TaqMan® MicroRNA Assays
miRNA Profiling: Save Time and Sample Using Real-Time PCR

Streamlined workflow enables miRNA profiling using real-time RT-PCR as an alternative to microarrays:

• Achieve wide dynamic range (TaqMan® Assay: 6–7 logs vs. microarrays: 2–3 logs)
• Use low sample input (TaqMan Assay: 160–320 ng vs. microarrays: 1–5 µg)
• Follow simple workflow —45-fold fewer steps than singleplex RT and faster workflow than arrays (TaqMan Assay: 3 hours vs. microarrays: 1–2 days)
• Cut RT enzyme costs 6 fold
• Use Multiplex RT pools with individual TaqMan MicroRNA Assays or TaqMan Low Density Array Human MicroRNA Panel v1.0

miRNA Profiling
MicroRNAs (miRNAs) are small RNA molecules that regulate cellular processes by affecting the translation or stability of target mRNA. More than one third of human genes may be regulated by miRNAs and each miRNA is believed to regulate multiple genes. miRNA profiling by microarray analysis has been used to correlate miRNAs with cell processes and disease and to establish their use as potential biomarkers. Microarrays make it possible to spot many targets (and multiple species) on a single array, allowing them to be analyzed affordably. However, the microarray workflow is cumbersome, the sample requirements are significant, and the sensitivity and specificity are limited. Applied Biosystems TaqMan MicroRNA Assays have been used by researchers to validate miRNA profiling data generated on microarray platforms, taking advantage of the increased sensitivity, specificity, and dynamic range of these real-time PCR methods. Now they can be used for the initial profiling itself.

Using Real-time-PCR for miRNA Profiling
Applied Biosystems Multiplex RT pools provide an approach for human miRNA profiling that uses real-time RT-PCR. They are a set of multiplexed reverse transcription primer pools that streamline the workflow for TaqMan chemistry-based miRNA profiling. This alternative to microarrays provides significant advantages in data quality (i.e., higher sensitivity to low expressors; greater specificity for the mature, biologically active form; and increased assay dynamic range necessary for the broad range of miRNA expression) and in the process saves money, time, and sample (Figure 1). Each pool comprises up to 48 RT primers, including two endogenous controls provided for data normalization. The primer pools were created using state-of-the-art bioinformatic analysis and have been confirmed by wet-chemistry testing to ensure that there are no deleterious primer:primer interactions. The primers included in each pool are identical to those available for individual miRNA RT-PCR (TaqMan MicroRNA Assays), enabling miRNAs of interest to be followed up using the individual assays.

Figure 1. Save 45 Steps Using Multiplex RT Pools Workflow. Multiplex RT pools can be used in place of individual MicroRNA RT primers (singleplex RT) to increase throughput for miRNA analysis, decreasing the workflow by 45 steps.This makes miRNA expression profiling possible using TaqMan® chemistry, while enabling increased speed and additional sensitivity over array expression profiling methods. Note that Multiplex RT pools can be used with TaqMan Low Density Arrays for a further enhancement to the workflow.

Only 8 RT reactions are required to obtain coverage of 365 human miRNAs and two endogenous control genes using the complete set of Multiplex RT pools. This is in contrast to the 300–400 individual miRNA RT reactions that would otherwise be required, and results in tremendous time and cost savings.

Faster Profiling with Less Sample: Just 3 hr and 200 ng for a Human miRNA Profile
In combination with the TaqMan MicroRNA Assays, the Multiplex RT pools provides researchers an affordable human miRNA profiling method that is 6 fold faster, requires 3–10 fold less sample, and provides higher data quality than microarray-based methods. The streamlined experimental workflow reduces the number of RT reactions approximately 45-fold and the amount of input sample required by 2–10 fold (Figure 2) while lowering the cost of running TaqMan MicroRNA Assays, making it an ideal tool for miRNA profiling.

Figure 2. Sample Input Requirements for miRNA Screening with TaqMan® MicroRNA Assays (365 miRNA targets + 2 endogenous control genes).

What you Get
Designed for use with the TaqMan MicroRNA Assays, each Multiplex RT pool is provided in a separate 0.5 mL screwcap, skirted tube for purchase individually or as a full set. Each 10X tube contains enough Multiplex RT reagent for 50–100 samples (40 µL RT–20 µL RT reactions, respectively).