Personally, I tested the expression of seven different housekeeping genes in two different cell lines across five different experimental treatments ( Figure 1). Keep in mind this will likely be different across different cell lines too! How Do I Choose the Right Housekeeping Gene? In fact, none of these genes were present in the top 50 most stably expressed genes in one study! To confidently be able to ensure consistent and correct results, you need a housekeeping gene whose expression is stable across all of the parameters you are assessing. Common housekeeping genes such as beta-actin, HPRT, GAPDH and 18S have all been shown to have unstable expression under different conditions and disease states, suggesting that they may not be suitable for use in all situations. The definition of a good housekeeping gene, is one that has stable expression in varying conditions, thus can be used to normalize other influences which may skew the result. Thus, a good housekeeping gene provides a point of reference to normalize all values to and ensure that the effect of varying DNA content is minimized. Owing to the sensitivity of qRT-PCR, slight differences in the DNA content of samples caused by pipetting errors, inefficient cDNA synthesis and inaccurate quantification will lead to a quantifiable difference. To be able to compare the expression level, you need to ensure that their initial input was the same, because qRT-PCR will exponentially increase your desired product. Unless you are assessing absolute quantification by generating a standard curve, you generally are assessing relative expression (the difference between two samples). First, you need to test your primers for specificity and efficiency and second, and most commonly overlooked, you need to optimize and select your housekeeping gene. Want to measure how much mRNA you have in a particular sample? Easy! Make some cDNA, add some fluorescent DNA-intercalating dye, pop it into a quantitative real-time PCR (qRT-PCR) machine and Bob’s your uncle! You have your result! Easy right…? Not so fast.Īs with any scientific assay, qRT-PCR requires some optimization.
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