High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA

Abstract

MicroRNAs (miRNAs) are an emerging class of small non-coding RNAs implicated in a wide variety of cellular processes. Research in this field is accelerating, and the growing number of miRNAs emphasizes the need for high-throughput and sensitive detection methods. Here we present the successful evaluation of the Megaplex reverse transcription format of the stem-loop primer-based real-time quantitative polymerase chain reaction (RT-qPCR) approach to quantify miRNA expression. The Megaplex reaction provides simultaneous reverse transcription of 450 mature miRNAs, ensuring high-throughput detection. Further, the introduction of a complementary DNA pre-amplification step significantly reduces the amount of input RNA needed, even down to single-cell level. To evaluate possible pre-amplification bias, we compared the expression of 384 miRNAs in three different cancer cell lines with Megaplex RT, with or without an additional pre-amplification step. The normalized Cq values of all three sample pairs showed a good correlation with maintenance of differential miRNA expression between the cell lines. Moreover, pre-amplification using 10 ng of input RNA enabled the detection of miRNAs that were undetectable when using Megaplex alone with 400 ng of input RNA. The high specificity of RT-qPCR together with a superior sensitivity makes this approach the method of choice for high-throughput miRNA expression profiling.

Figure 1.

Correlation plot between Cq values obtained through Megaplex RT alone (Cq_NP) and Megaplex RT with pre-amplification (Cq_P) using 10 ng of total RNA (A) and 1 ng of total RNA (B) from NGP neuroblastoma cells. (A) Data points highlighted in red indicate miRNAs that are only detectable if pre-amplification is applied. (B) Data points highlighted in red indicate miRNAs that are only detectable if no pre-amplification is applied.

Figure 2.

Cq–Cq correlation plot for two independent reverse transcription reactions without pre-amplification.

Figure 3.

Difference in differential miRNA expression (ΔΔCq) between two neuroblastoma cell lines (NBL-S and IMR-32) as measured using Megaplex RT alone (ΔCq_NP) and Megaplex RT with pre-amplification (ΔCq_P). ΔΔCq values are plotted in function of the average expression of each miRNA in the two cell lines as quantified by Megaplex RT alone (average Cq_NP). Bar plots indicate the mean ΔΔCq value for miRNAs with an average Cq_NP value ranging between 15–20, 20–25, 25–30 and 30–35. (A) Results when a Cq detection cutoff of 35 is applied. (B) Results when Cq detection cutoff is lowered to 30.

Figure 4.

(A) Standard curve analysis depicting perfect linearity and correlation between input cell number (1–128) and measured Cq value for three different miRNAs (miR-18a, miR-20b and miR-106a). (B) Standard curve analysis depicting perfect linearity and correlation between total RNA input (2–6250 pg) and measured Cq value for three different miRNAs (miR-92, miR-19a and miR-20a). Total input RNA values on X-axis are log₂ based.