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Simple, Rapid, Quantitative, and Sensitive Tools for miRNA Profiling
Developing profiling strategies that are efficient and quantitative has proven to be challenging for microRNA (miRNA) because of their small sizes and the sequence similarity among family members. TaqMan® MicroRNA Assays are highly sensitive and specific assays that are now available pre-loaded in the TaqMan Array Human MicroRNA Panel, a 384-well micro-fluidic card. Coupled with miRNA Multiplex RT pools, the Human MicroRNA Panel provides a simple, rapid, quantitative, and sensitive tool for miRNA profiling.
Design and Workflow
Multiplexing the reverse transcription (RT) primer is necessary when screening large numbers of miRNAs. The eight Multiplex RT pools for TaqMan MicroRNA Assays each provide a 48-plex RT primer pool that contains up to 46 unique miRNA RT primers and two endogenous controls (RNU48 and RNU44) for data normalization. These multiplexed RT reactions can be used in conjunction with the TaqMan Array Human MicroRNA Panel to rapidly screen 365 human miRNAs in as few as 3 hours.
In addition to the 365 unique human TaqMan MicroRNA Assays, the Human MicroRNA Panel contains 17 human endogenous control assays (8 replicates each of the RNU48 and RNU44 control assays and a single RNU6B control assay). All of the assays are pre-loaded into a 384-well micro-fluidic card that has 8 sample-loading ports. Each port is connected to 48 reaction wells that contain up to 46 TaqMan MicroRNA Assays and 2 endogenous controls that correspond to one of the eight Multiplex RT reactions.
The workflow for using the Human MicroRNA Panel for miRNA profiling is simple and fast (Figure 1). First, RNA is converted to cDNA using the Multiplex RT and TaqMan MicroRNA Reverse Transcription Kit (i.e., eight 48-plex RT reactions per sample). The cDNA samples are then diluted and mixed with TaqMan Universal PCR Master Mix and pipetted into its corresponding sample-loading port on the micro-fluidic card. After a brief centrifugation, the Human MicroRNA Panel is sealed and is ready to run on the Applied Biosystems 7900HT Fast Real-Time PCR System. The whole procedure from cDNA to running TaqMan reactions takes only a few minutes and does not require robotics.
In addition to the 365 unique human TaqMan MicroRNA Assays, the Human MicroRNA Panel contains 17 human endogenous control assays (8 replicates each of the RNU48 and RNU44 control assays and a single RNU6B control assay). All of the assays are pre-loaded into a 384-well micro-fluidic card that has 8 sample-loading ports. Each port is connected to 48 reaction wells that contain up to 46 TaqMan MicroRNA Assays and 2 endogenous controls that correspond to one of the eight Multiplex RT reactions.
The workflow for using the Human MicroRNA Panel for miRNA profiling is simple and fast (Figure 1). First, RNA is converted to cDNA using the Multiplex RT and TaqMan MicroRNA Reverse Transcription Kit (i.e., eight 48-plex RT reactions per sample). The cDNA samples are then diluted and mixed with TaqMan Universal PCR Master Mix and pipetted into its corresponding sample-loading port on the micro-fluidic card. After a brief centrifugation, the Human MicroRNA Panel is sealed and is ready to run on the Applied Biosystems 7900HT Fast Real-Time PCR System. The whole procedure from cDNA to running TaqMan reactions takes only a few minutes and does not require robotics.
Figure 1. Workflow Using the Multiplex RT and TaqMan® Array Human MicroRNA Panel
High Sensitivity and Wide Dynamic Range
The RNA sample used here consisted of a pool of Ambion FirstChoice® Total RNA from heart, brain, lung, placenta, testis, spleen, and ovary. This total RNA pool was combined with a pool of synthetic RNA oligonucleotides corresponding to all miRNA targets interrogated by the Multiplex RT and Human MicroRNA Panel.
The sensitivity and dynamic range of the Human MicroRNA Panel is demonstrated by a titration experiment in which 10-fold serial dilutions of RNA samples were analyzed. The RNA sample at 10X (10 ng/µL total RNA pool and 100 pM of each synthetic target), 1X, 0.1X, and 0.01X concentration was reverse transcribed using each of the eight Multiplex RT primer pools and Applied Biosystems MicroRNA Reverse Transcription Kit. A no-template-control (NTC) was also included to examine the specificity of the miRNA assays.
The Human MicroRNA Panel is highly sensitive: all assays showed detectable signals (CT <35) at 10X and 1X RNA input concentrations, while 99.7% and 88% detection were observed at lower RNA input concentrations of 0.1X and 0.01X, respectively (~100 fM per target) (Figure 2A). In addition, all assays had NTC signals of CT >37, demonstrating that these assays were very specific. The Human MicroRNA Panel also showed good linear response with a median R2 value of 0.97 for all the assays across 3 RNA concentrations tested (Figure 2B).
Figure 2. Sensitivity and Dynamic Range of TaqMan® Array Human MicroRNA Panel. (A) Box plot of the distribution of CT values for five RNA input concentrations: NTC (no template control), 0.01X, 0.1X, 1X, and 10X. Brown triangle=median CT value (B) Dynamic range of representative miRNA. The R2 values were calculated from a linear regression fit of the CT values derived from four RNA input concentrations (orange line).
The sensitivity and dynamic range of the Human MicroRNA Panel is demonstrated by a titration experiment in which 10-fold serial dilutions of RNA samples were analyzed. The RNA sample at 10X (10 ng/µL total RNA pool and 100 pM of each synthetic target), 1X, 0.1X, and 0.01X concentration was reverse transcribed using each of the eight Multiplex RT primer pools and Applied Biosystems MicroRNA Reverse Transcription Kit. A no-template-control (NTC) was also included to examine the specificity of the miRNA assays.
The Human MicroRNA Panel is highly sensitive: all assays showed detectable signals (CT <35) at 10X and 1X RNA input concentrations, while 99.7% and 88% detection were observed at lower RNA input concentrations of 0.1X and 0.01X, respectively (~100 fM per target) (Figure 2A). In addition, all assays had NTC signals of CT >37, demonstrating that these assays were very specific. The Human MicroRNA Panel also showed good linear response with a median R2 value of 0.97 for all the assays across 3 RNA concentrations tested (Figure 2B).
Figure 2. Sensitivity and Dynamic Range of TaqMan® Array Human MicroRNA Panel. (A) Box plot of the distribution of CT values for five RNA input concentrations: NTC (no template control), 0.01X, 0.1X, 1X, and 10X. Brown triangle=median CT value (B) Dynamic range of representative miRNA. The R2 values were calculated from a linear regression fit of the CT values derived from four RNA input concentrations (orange line).
High Reproducibility
As described earlier, two endogenous controls, RNU48 and RNU44, are included in each of the eight 48-plex Multiplex RT pools and replicated on the Human MicroRNA Panel eight times (one replicate in each port). The intra-array reproducibility of the Human MicroRNA Panel was assessed by determining the standard deviation of the CT measurements of these replicated endogenous controls. The standard deviation of RNU48 was consistently under 0.5 across 3 logs of RNA input concentrations (Figure 3A). Of note, the results for RNU48 were obtained from cDNA independently prepared from eight Multiplex RT pools that have different primer compositions. To evaluate the inter-array reproducibility, cDNA generated from Multiplex RT reactions were run on two Human MicroRNA Panels. A scatter plot of the measurements from the two replicates is shown in Figure 3B. A linear regression fitting of all data showed a slope of 0.992 and an R2 value of 0.999, demonstrating that the Human MicroRNA Panel is highly reproducible between arrays.
Figure 3. Reproducibility of TaqMan® Array Human MicroRNA Panel. (A) Intra-array reproducibility in four RNA input concentrations across 3 logs of dynamic range. Box plot summarized the CT distribution, mean CT value, and standard deviation of the eight replicates of RNU48 (endogenous control) represented on each TaqMan Human MicroRNA Panel. (B) Inter-array reproducibility. Scatter plot of CT values measured by two TaqMan Human MicroRNA Panels. Data were derived from two experiments (with two RNA input concentrations: 1X and 10X), and 764 data points were represented.
Figure 3. Reproducibility of TaqMan® Array Human MicroRNA Panel. (A) Intra-array reproducibility in four RNA input concentrations across 3 logs of dynamic range. Box plot summarized the CT distribution, mean CT value, and standard deviation of the eight replicates of RNU48 (endogenous control) represented on each TaqMan Human MicroRNA Panel. (B) Inter-array reproducibility. Scatter plot of CT values measured by two TaqMan Human MicroRNA Panels. Data were derived from two experiments (with two RNA input concentrations: 1X and 10X), and 764 data points were represented.
Concordance of Array Results with Individual Assay Results
Figure 4 is a scatter plot of CT values measured by the Human MicroRNA Panel and individual TaqMan MicroRNA Assays, using four RNA input concentrations across three logs of dynamic range. A linear regression fitting of the data showed a slope of 1.001 and an R2 value of 0.990. These results demonstrate that the miRNA expression measurements by the Human MicroRNA Panel and individual TaqMan MicroRNA Assays are highly concordant.
Figure 4. RNA Input Concentrations Scatter Plot of CT Values Measured by TaqMan® Human MicroRNA Panel and by Individual TaqMan MicroRNA Assays. 1376 data points, derived from four experiments (with four RNA input concentrations across 3 logs of dynamic range), are represented in this plot. A linear regression fitting and the corresponding R2 value are presented.
Figure 4. RNA Input Concentrations Scatter Plot of CT Values Measured by TaqMan® Human MicroRNA Panel and by Individual TaqMan MicroRNA Assays. 1376 data points, derived from four experiments (with four RNA input concentrations across 3 logs of dynamic range), are represented in this plot. A linear regression fitting and the corresponding R2 value are presented.
Conclusions
The TaqMan Array Human MicroRNA Panel, when used in conjunction with Multiplex RT pools, provides a simple, rapid, and highly quantitative tool for miRNA profiling. The high concordance with individual TaqMan MicroRNA Assays, linearity over the dynamic range of the sample, reproducibility, and sensitivity, as well as considerable time and sample savings, all mean that these two products provide a competitive method for microarray screening.
Scientific Contributors
Yulei Wang, Kathy Lee, Iain Russell, Carolyn Gonzalez, Yu Liang, Caifu Chen • Applied Biosystems, Foster City, CA
Scientific Contributors
Yulei Wang, Kathy Lee, Iain Russell, Carolyn Gonzalez, Yu Liang, Caifu Chen • Applied Biosystems, Foster City, CA