Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool 
in profiling gene expression levels. One of its many advantages is a lower 
detection limit compared to other methods of gene expression profiling while 
using smaller amounts of input for each assay. Automated qPCR setup has improved 
this field by allowing for greater reproducibility. Its convenient and rapid 
setup allows for high-throughput experiments, enabling the profiling of many 
different genes simultaneously in each experiment. This method along with 
internal plate controls also reduces experimental variables common to other 
techniques. We recently developed a qPCR assay for profiling of pre-microRNAs 
(pre-miRNAs) using a set of 186 primer pairs. MicroRNAs have emerged as a novel 
class of small, non-coding RNAs with the ability to regulate many mRNA targets 
at the post-transcriptional level. These small RNAs are first transcribed by RNA 
polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved 
into the precursor miRNA (pre-miRNA). Pre-miRNAs are exported to the cytoplasm 
where Dicer cleaves the hairpin loop to yield mature miRNAs. Increases in miRNA 
levels can be observed at both the precursor and mature miRNA levels and 
profiling of both of these forms can be useful. There are several commercially 
available assays for mature miRNAs; however, their high cost may deter 
researchers from this profiling technique. Here, we discuss a cost-effective, 
reliable, SYBR-based qPCR method of profiling pre-miRNAs. Changes in pre-miRNA 
levels often reflect mature miRNA changes and can be a useful indicator of 
mature miRNA expression. However, simultaneous profiling of both pre-miRNAs and 
mature miRNAs may be optimal as they can contribute nonredundant information and 
provide insight into microRNA processing. Furthermore, the technique described 
here can be expanded to encompass the profiling of other library sets for 
specific pathways or pathogens.