Analysis of non-specific amplification in stem-loop primer RT-qPCR

The stem-loop RT-qPCR commonly used for miRNA detection is prone to non-specific amplification. To clarify the source of non-specific amplification and characterize its sequence features, we sequenced the non-specific amplification products. Next-generation sequencing of amplification products identified sequence mismatches between non-specific and positive controls, consistent with primer-primer interactions between the stem-loop primer and upstream primer 3' regions.