Comprehensive molecular screening: from the RT-PCR to the RNA-seq.

Up to now, the analysis of the mRNA expression in tumoral and non-tumoral has been conducted via RT-PCR. It is considered to be the gold standard for measuring the number of copies of specific cDNA targets. The application of RT-PCR has demonstrated that levels of RNA transcripts stratify patients and predict outcomes in a variety of diseases, providing the basis for several important clinical tests. However, the inherent variability in the quality of any quantitative PCR data makes it difficult to replicate and the analysis is time consuming in the laboratory for the analysis of one gene. Moreover, comparing expression levels across different experiments is often difficult and can require complicated normalization methods. Many techniques have been developed over the years but without good clinical applications. A new, simple and effective way to measure transcriptome composition and to discover new exons or genes is by the RNA-seq. Some advantages of this technique are high reproducibility, the large dynamic range, requirement of less sample RNA, and the ability to detect novel transcripts, alternative splicing, even in the absence of a sequenced genome. However, this RNA-Seq technique will not likely replace current RT-PCR methods, but will be complementary depending on the needs and the resources of the clinic and the laboratory as the results of the RNA-Seq will identify those genes that need to then be examined using RT-PCR methods. The application of the two complementary technologies in the routine analysis of cancer laboratories would be useful in characterizing patients and would assist oncologists in making clinical decisions, as it allows us to identify all molecular characteristics of the tumor.