Following the reverse transcription a method for measuring the amount of cDNA that has been reverse transcribed would be desirable; the only reliable method to do this is to quantify the synthesis of radio labeled cDNA. This strategy was more commonly used 10 years ago but the requirement for using radio isotopes coupled with the extensive increase in the use of qRT-PCR has resulted in this method rarely being used today.
Alternative methods have been proposed using spectrophotometry or DNA specific binding dyes but the major problem is that there is usually much more RNA than cDNA present after a reverse transcription. However, these are not satisfactory, as the background noise levels generate additional errors that tend to mask our ability to quantitate accurately the amount of synthesized cDNA. However, new methods for more accurate, non-radioactive quantification of cDNA synthesis efficiency are being developed and it is likely that normalization against cDNA will become feasible in the not too distant future.
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