Once the RNA has been extracted, the next stage of normalization can be performed by ensuring that the same amount is used for each reverse transcription reaction. Not only does this facilitate normalization but circumvents problems associated with the linearity of the reverse transcrip-tase step. However, normalizing against total RNA requires accurate measurement of RNA quantity and again this is not straightforward. A number of different methods are available; most commonly, optical methods using spectrophotometry or fluorimetry with fluorescent dyes (Ribogreen) are used and provide varying degrees of accuracy. What is also important, but often overlooked, is the need to assess the quality of RNA because degraded RNAs may adversely affect results (Bustin and Nolan, 2004). Most commonly, gel electrophoresis or analysis using the Agilent Bioanalyser/Biorad Experion is used to determine the integrity of the ribosomal RNA (rRNA) band.
There are several drawbacks to normalization against total RNA: it is not easy to take into consideration differences in mRNA integrity, it does not allow for differences caused by the presence of inhibitors and ignores the efficiency of converting RNA into cDNA. Even if RNA integrity is assessed, any quality statement refers to rRNA, not mRNA. Total RNA comprises ~80% rRNA (with the protein coding mRNA comprising ~2-5% depending cell type); consequently, degradation of rRNA may not directly reflect degradation of mRNA. In addition, the relative amounts of rRNA and mRNA often differ between samples, especially when the mRNA originates from highly proliferating tissue, e.g., cancers. Of course, it is possible to extract mRNA, but this involves additional steps thereby potentially increasing experimental error, which can significantly reduce yield and may result in long-term stability problems. Furthermore, mRNA extraction is currently limited to the extraction of poly-adenylated mRNAs so will not purify non-adenylated mRNAs, for example those specifying histones.
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