Comparison of RiboGreen (R) and 18S rRNA quantitation for normalizing real-time RT-PCR expression analysis

摘要

Real-time RT-PCR is becoming the method of choice for monitoring or confirming mRNA expression. With unparalleled sensitivity, the ability to use 100 to 1000 times less RNA than other methodologies, and high-throughput potential for gene discovery approaches, real-time RT-PCR gives researchers the ability to determine mRNA abundance quickly and efficiently. While Northern blot analysis remains the most accepted method for measuring mRNA abundance, there are drawbacks that limit its utility. First, Northern blot analysis requires several micrograms of total RNA for each lane, which precludes the analysis of microdissected samples, tissue from laser capture, or other valuable samples due to the high cost and/or inability to obtain sufficient amounts of RNA. Second, Northern analysis is limited by the number of samples that can be run simultaneously on a gel. or requires standard or control samples on each blot for normalization between other Northern blots. Finally, the limited sensitivity of Northern analysis, given the indirect nature of the measurement and the multiple steps required, necessitates a robust change in the abundance of a given transcript to obtain a statistically significant result.