Quantitation of microRNAs by real-time RT-qPCR.

MicroRNAs (miRNAs) are ∼22 nucleotide regulatory RNA molecules that play important roles in controlling developmental and physiological processes in animals and plants. Measuring the level of miRNA expression is a critical step in methods that study the regulation of biological functions and that use miRNA profiles as diagnostic markers for cancer and other diseases. Even though the quantitation of these small miRNA molecules by RT-qPCR is challenging because of their short length and sequence similarity, a number of quantitative RT-qPCR-based miRNA quantitation methods have been introduced since 2004. The most commonly used methods are stem-loop reverse transcription (RT)-based TaqMan(®) MicroRNA assays and arrays. The high sensitivity and specificity, large dynamic range, and simple work flow of TaqMan(®) MicroRNA assays and arrays have made TaqMan analysis the method of choice for miRNA expression profiling and follow-up validation. Other methods such as poly (A) tailing-based and direct RT-based SYBR miRNA assays are also discussed in this chapter.

Asynchronous PCR.

Asynchronous PCR (aPCR) is a new PCR method that directs an ordered and sequential amplification of the + and - strands of DNA amplicons. There are several unique characteristics of aPCR that generate new application opportunities. The melting temperature (Tm) of the forward and reverse aPCR primers differ by at least 15°C. The concentration of the lower Tm primer is reduced from 900 to 100 nM, thereby allowing for asynchronous or asymmetric strand-specific amplification. Furthermore, unique thermocycling parameter strategy dictates the + and - strand amplification cue. Each aPCR cycle includes two annealing and extension steps. Sequential annealing and extension of forward and reverse primers during each cycle produce transient single-stranded DNA (ssDNA) amplicons which help hybridization-based probes such as peptide nucleic acid (PNA) bind to the target sequences more effectively. This new method can be used in real-time quantitative PCR (qPCR) for gene expression analyses as well as production of robust ssDNA targets for microarray and other hybridization-based applications.