How important is the RNA purification process, for obtaining reliable qRT-PCR results?
The most important prerequisite for any gene expression analysis experiment is the preparation of consistent, high-quality RNA from every experimental sample. Contamination by DNA, protein, polysaccharide, or organic solvents can jeopardize the success of an experiment. Genomic DNA contamination in an RNA sample compromises the quality of gene expression analysis results. The contaminating DNA inflates the OD reading of the RNA concentration. It also is a source of false positive signals in RT-PCR experiments. RNase contamination degrades your RNA samples, causes low signal and false negative results in the PCR. Residual polysaccharides, collagen, other macromolecules or organic solvents in an RNA sample can inhibit the activity of DNase, which may interfere with DNase treatment for genomic DNA removal. These contaminants may also inhibit reverse transcriptase and DNA polymerase, leading to lower reverse transcription efficiency and reduced PCR sensitivity.
