mRNA Identification and Quantification

Molecular technologies are currently evolving rapidly the biological sciences. This results in an immense progress in accumulation of new data potentially useful for molecular diagnostics. One of these highly sophisticated methodologies is the quantitative assessment of target nucleic acids, mostly performed as quantitative polymerase chain reaction (PCR) on DNA level or combined with reverse transcription PCR (RT-PCR) to investigate the transcriptome on RNA level. To quantify local tissue-specific expression even in tissues with low abundances, very sensitive methods are required, which allow reliable mRNA quantification. The aim of a full quantitative method is to estimate, as exact and reliable as possible, the number or target molecules in the sample. For this exact quantification of low abundant gene expression only a few PCR methods allow reliable mRNA quantification. (A) Internally standardized competitive RT-PCR measured by high-performance liquid chromatography separation and ultraviolet detection or high-resolution gel electrophoresis followed by densitometric analysis: in a competitive RT-PCR, a reference RNA mutant is reverse transcribed and coamplified in the same reaction tube with the native mRNA sequence of interest. Internally standardized RT-PCR is a very time consuming and laborious technique. It is generally believed to yield the most precise results, because all parameters throughout RT-PCR act on both the analyte and reference mutant. (B) Externally standardized RT-PCR with online-detection using SYBR Green I technology: real-time RT-PCR with SYBR Green I detection produces sensitive and reliable results. Due to the use of an external standard curve, the amplification efficiencies for the calibration curve and the analyte....