Specific and sensitive quantitative RT-PCR of miRNAs with DNA primers

Abstract

Background: MicroRNAs are important regulators of gene expression at the post-transcriptional level and play an important role in many biological processes. Due to the important biological role it is of great interest to quantitatively determine their expression level in different biological settings.

Results: We describe a PCR method for quantification of microRNAs based on a single reverse transcription reaction for all microRNAs combined with real-time PCR with two, microRNA-specific DNA primers. Primer annealing temperatures were optimized by adding a DNA tail to the primers and could be designed with a success rate of 94%. The method was able to quantify synthetic templates over eight orders of magnitude and readily discriminated between microRNAs with single nucleotide differences. Importantly, PCR with DNA primers yielded significantly higher amplification efficiencies of biological samples than a similar method based on locked nucleic acids-spiked primers, which is in agreement with the observation that locked nucleic acid interferes with efficient amplification of short templates. The higher amplification efficiency of DNA primers translates into higher sensitivity and precision in microRNA quantification.

Conclusions: MiR-specific quantitative RT-PCR with DNA primers is a highly specific, sensitive and accurate method for microRNA quantification.

Figure 1

Flow scheme of miR-specific qPCR. 1. Start with purified RNA containing miRNA. 2. Add poly(A) tail with poly(A) polymerase (PAP). 3. cDNA synthesis with reverse transcriptase (RTase) and an anchored poly(T) primer with a 5' tag. 4. PCR with two tagged primers.

Figure 2

MiR-specific qPCR on synthetic templates with DNA primers. A The effect of primer concentration on Cq value of ssc-let-7d and ssc-miR-26a miR-specific qPCR assays. Real-time PCR assays were performed in parallel at three different concentrations (125, 250 and 500 nM) of the forward and of the reverse primers. B Amplification curves of an eight log₁₀ dilution series of a synthetic ssc-let-7d template in the ssc-let-7d miR-specific qPCR assays. All samples contained a final concentration of 0.2 ng/μl salmon sperm DNA. C Extrapolation of Cq as function of the log₁₀ of the number of templates for the same experiment as in B was a straight line (R² = 0.9993) with slope of -3.341 (PCR efficiency = 99%) over eight log₁₀ dilution of the template. D Melting curve analysis of the same experiment. No template control is labeled ntc. Melting curve analysis was performed from 60°C to 99°C.

Figure 3

MiR-specific qPCR on biological samples with DNA primers. A Amplification curves of 40 uterus samples with the ssc-miR-150 miR-specific qPCR assay. B Melting curve analysis of the same experiment. Melting curve analysis was performed from 55°C to 95°C. C Extrapolation of Cq as function of the log₁₀ of the number of templates for the same experiment as in A was a straight line (R² = 1.0) with a slope of -3.406 (PCR efficiency = 97%) over 4 log₁₀ dilution of a pool that includes all samples included in the study.

Figure 4

Discrimination between miRNAs with single nucleotide differences. A Position of the single nucleotide mismatches relative to the PCR primers for the ssc-let-7a, ssc-miR-23a, ssc-miR-125b and ssc-miR-150 qPCR assays. The ssc-miR-23b sequence used for mismatch discrimination was taken from miRBase and is different from the ssc-miR-23b sequence found in uterus and used for designing the ssc-miR-23b qPCR primers (Table 1). B Discrimination between closely related miRNA templates for miR-specific qPCR assays with DNA primers. Mismatches in the miRNA compared to the PCR primers are underlined. The data represents the results of three to four measurements. C Amplification curves of ssc-let-7a and ssc-let-7e synthetic template in the ssc-let-7a miR-specific qPCR assays. All samples including the no template control (ntc) contained a final concentration of 0.2 ng/μl salmon sperm DNA.

Figure 5

MiR-specific qPCR in different qPCR master mixes. A Comparison of amplification curves of a synthetic ssc-let-7d template in the ssc-let-7d miR-specific qPCR assay in QuantiFast and in Brilliant III qPCR Master mixes. B Melting curve analysis of the same experiment. No template control is labeled ntc. Melting curve analysis was performed from 60°C to 99°C. No change in fluorescence (dF/dT = 0) was observed above 80°C and this part of the curves was omitted from the figure. C Extrapolation of Cq as function of the log₁₀ of the number of templates for the same experiment as in A was a straight line (R² indicated on figure) and for both master mixes the PCR efficiency was 99% as calculated from the slope of the regression line.