A poly(A) tail is added to the mature miRNA template (step 1A). cDNA is synthesized using a Poly(T) primer with a 3’ degenerate anchor and a 5’ universal tag (step 1B). The cDNA template is then amplified using two miRNA-specific and LNA-enhanced forward and reverse primers (step 2A). SYBR Green is used for detection (step 2B).
Data from serial dilutions of human AM6000 total RNA are shown. All miRNA assays exhibit linear readout with correlation coefficients R2 > 0.99.
A 10-fold serial dilution (ranging from 1 x 108 to 10 copies) of cDNA template made from synthetic hsa-miR-181a was used for real-time PCR amplification. The standard curve shows excellent linear correlation (R2 = 0.998) between the decreasing cycle numbers and the logarithm of the miRNA copy number. The assay can detect as few as 10 miRNA copies in the PCR reaction and has a dynamic range of 8 logs. Efficiency calculated from the standard curve = 1.94. The dilution series was performed in a background of MS2 bacteriophage total RNA.
The miRCURY LNA miRNA PCR System uses one single cDNA synthesis reaction for all amplifications, reducing pipetting and saving time and sample. Two LNA-enhanced miRNA-specific qPCR primers enable highly specific and sensitive amplification and single nucleotide discrimination. The fast and easy workflow takes only 3 hours with minimal hands-on steps.
Specificity was assessed using 8 pools each containing at least 80 different synthetic miRNAs. Closely related miRNAs are placed in different pools. This example shows results from the hsa-miR-1 assay. The miRCURY LNA miRNA PCR System performs perfectly with a high signal in pool 1, which contains the hsa-miR-1 synthetic miRNA target, and only a borderline signal in pool 7, which contains the most closely related miRNA (hsa-miR-206). The competitor platform uses ligation-based cDNA synthesis and only one miRNA-specific probe. Competitor T shows a lack of specificity. The highest signal for Competitor T is obtained with pool 1. However, non-specific false-positive signals are obtained from all other pools in the absence of hsa-miR-1 template, within a Cq range that would be interpreted as real signals.
miRCURY LNA miRNA PCR Primers and commercially available DNA-based primer sets were used for real-time PCR amplification of serially diluted cDNA (106–10 copies). LNA-based primer sets showed superior sensitivity in all cases, especially for AT-rich sequences, such as hsa-miR-155 (61% AT) and hsa-miR-1 (73% AT).
Areas between 8000 and 13000 μm2 of normal tissue, tumor tissue and tumor stroma were isolated by laser dissection from FFPE sections of human colon (A). Total RNA was extracted and miRNA levels were quantified using miRCURY LNA miRNA PCR (B). Normalized data are shown on a log2 scale as relative expression compared to normal tissue. hsa-miR-103 and hsa-let-7a were used as reference genes.
Normalized expression levels of eight different miRNAs in two serum samples are shown. Total RNA purified from the equivalent of 8 µl serum was used in the RT reaction. hsa-let-7a and hsa-miR-103 were used as reference genes for normalization.
Amplification curves (A) and melting curves (B) from the hsa-miR-145 miRCURY LNA miRNA PCR Assay performed on a serial dilution of human colon total RNA (100 ng to 1 pg), performed in triplicate. The amplification curves demonstrate sensitive and reproducible detection down to 1 pg of total RNA input with no background signal. The melting curves show only one major Tm peak corresponding to a well-defined melting temperature of the amplicon, demonstrating specific amplification of hsa-miR-145.
The LNA-enhanced, miRNA-specific primers facilitate the design of miRNA qPCR assays that discriminate between closely related miRNA sequences. The table shows the percent signal obtained from target with a single nucleotide mismatch compared to the signal from the perfectly matched target (set to 100%). Single nucleotide discrimination is possible at various positions in the miRNA sequence.