Journal
ANALYTICAL BIOCHEMISTRY
Volume 342, Issue 1, Pages 69-77Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2005.03.034
Keywords
breast cancer; cancer progression; real-time RT-PCR; housekeeping genes; internal standard
Funding
- NCI NIH HHS [R01 CA77575, R01 CA80130, R24 CA83148] Funding Source: Medline
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Real-time reverse transcriptase polymerase chain reaction is recognized as a highly sensitive and specific method for quantification of mRNA expression. SYBR green I dye simplifies the experimental design but introduces the need for specific controls to maintain high specificity. Due to this increased sensitivity, standards that may have been acceptable for normalization of less sensitive methods have been shown to vary considerably among cell lines, tissues, proliferative states, treatments, and developmental conditions and by degree of cancer progression. It has become evident that determination of suitable normalization standards is a requirement for the use of this method as it is applied toward any new experimental model. We have assessed the suitability of a number of commonly used standards for the normalization of mRNAs among a set of human breast cancer cell lines of increasing metastatic potential and have determined that I SS rRNA and beta-actin (A CTB) mRNA are both suitable for this purpose, with each having some limitations. 18S rRNA varies less among the cell lines but has a higher degree of random variability, while A CTB mRNA varies more among cell lines but has a lower degree of random variation. (c) 2005 Elsevier Inc. All rights reserved.
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