![]() ![]() Real-time PCR can also discriminate between messenger RNAs (mRNAs) with almost identical sequences, requires much less RNA template than other methods of gene expression analysis, and can be relatively high-throughput given the proper equipment. ![]() In addition, real-time PCR assays can reliably detect gene expression differences as small as 23% between samples ( 7) and have lower coefficients of variation (cv SYBR® Green at 14.2% TaqMan® at 24%) than end point assays such as band densitometry (44.9%) and probe hybridization (45.1%) ( 8). Real-time PCR assays are 10,000- to 100,000-fold more sensitive than RNase protection assays ( 4), 1000-fold more sensitive than dot blot hybridization ( 5), and can even detect a single copy of a specific transcript ( 6). It can produce quantitative data with an accurate dynamic range of 7 to 8 log orders of magnitude ( 3) and does not require post-amplification manipulation. There are many benefits of using real-time PCR over other methods to quantify gene expression. Consequently, the greater the quantity of target DNA in the starting material, the faster a significant increase in fluorescent signal will appear, yielding a lower C t ( 2). This value is usually referred to as cycle threshold (C t), the time at which fluorescence intensity is greater than background fluorescence. Reactions are characterized by the point in time (or PCR cycle) where the target amplification is first detected. This is achieved using a variety of different fluorescent chemistries that correlate PCR product concentration to fluorescence intensity ( 1). ![]() Real-time PCR is the technique of collecting data throughout the PCR process as it occurs, thus combining amplification and detection into a single step. The advent of real-time PCR and real-time reverse transcription PCR (real-time RT-PCR) has dramatically changed the field of measuring gene expression. ![]()
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