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2001. Muscle satellite cells are multipotential stem cells that exhibit myogenic, osteogenic, and adipogenic differentiation. Differentiation 68:245-253. Mechanisms of nascent fiber formation during avian skeletal muscle hypertrophy. 1999. Effect of turkey (Meleagridis gallopavo) breeder hen age and egg size on poult development. 2. Intestinal villus growth, enterocyte migration and proliferation of the turkey poult. Comp. Bio. Physio. Real-time RT-PCR is becoming the method of choice for monitoring or confirming mRNA expression (1−3). 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 (1,4). 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. All methods of RNA quantification rely on normalization methods to quantitatively compare multiple disparate samples. Due to the sensitivity of the assay, real-time RT-PCR requires accurate methods for normalizing both for the amount of RNA added to each reaction and for reaction efficiency (1,4). Standard methods used for nor-malization of RNA amounts include analysis of absorbance at 260 nm, or real-time RT-PCR analysis of housekeeping genes or 18S rRNA abundance (1). Absorbance at 260 nm is the most problematic approach because a large amount of RNA is required, samples will necessarily be subsequently diluted , and contaminates that are present in the sample frequently adversely influence the accuracy of the quantifica-tions. Housekeeping genes are suitable for normalization in some cases, but require additional analysis to verify the specific treatment or experimental conditions do not alter their steady-state mRNA abundance (1). The use of 18S rRNA abundance for normalization in real-time RT-PCR analysis has been effective in our hands. Advantages include the …

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

Comparison of RiboGreen^® and 18S rRNA quantitation for normalizing real-time RT-PCR expression analysis