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:: Posted by American Biotechnologist on 12-10-2009
Considering Real-Time PCR for gene expression analysis. Have you tested multiple reference genes or are you just going to run with your favorite such ast 18S? Check out this important article before you move forward. It could save you lots of grief in the long run.
Housekeeping genes are routinely used as endogenous references to account for experimental differences in gene expression assays. However, recent reports show that they could be de-regulated in different diseases, model animals, or even under varied experimental conditions, which may lead to unreliable results and consequently misinterpretations. This study focused on the selection of suitable reference genes for quantitative PCR in human hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) with different clinical outcomes.
:: Posted by American Biotechnologist on 11-12-2009
Bio-Rad Laboratories has put together a great tutorial on QPCR analysis including a thorough review of what the graphs mean and an overview of Absolute and Relative methods of quantification. The tutorial also includes a great explanation of the differences between Livak, delta CT and the Pfaffl methods of analyzing your results.
Watch part II: normalization against a reference gene
:: Posted by American Biotechnologist on 10-27-2009
There has been a lot of buzz lately on the newly published guidelines detailing the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines published by Bustin et al. in Clinical Chemistry 55:4, 2009. The paper was coauthored by several important PCR authorities including Jo Vandesompele and is therefore sure to become an important standard for real-time PCR publications. Dr. Sean Taylor of Bio-Rad has put together several short youtube videos explaining the importance of MIQE and how it can be applied to the bench.
:: Posted by American Biotechnologist on 10-16-2009
Nucleic acid amplification and detection are among the most valuable techniques used in biological research today. Scientists in all areas of research — basic science, biotechnology, medicine, forensic science, diagnostics, and more — rely on these methods for a wide range of applications. For some applications,
qualitative nucleic acid detection is sufficient. Other applications, however, demand a quantitative analysis. Choosing the best method for your application requires a broad knowledge of available technology.
This guide from Bio-Rad provides an introduction to many of the technical aspects of real-time PCR. It includes guidelines for designing the best real-time PCR assay for your experiments and explains how real-time PCR data are used in various applications.