ABI PRISM 7000 Sequence Detection Systems Relative ...
ABI PRISM 7000 Sequence Detection Systems Relative ...
ABI PRISM 7000 Sequence Detection Systems Relative ...
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Specifying the Components of an RQ Experiment<br />
Specifying the Components of an RQ Experiment<br />
RQ experiments require:<br />
• A target – The nucleic acid sequence that you are studying.<br />
• A calibrator – The sample used as the basis for comparative results.<br />
• An endogenous control – A gene present at a consistent expression level in all<br />
experimental samples. By using an endogenous control as an active reference, you<br />
can normalize quantification of a cDNA target for differences in the amount of<br />
cDNA added to each reaction. Note that<br />
– Each sample type (for example, each tissue in a study comparing multiple<br />
tissues) requires an endogenous control.<br />
– If samples are spread across multiple plates, each plate must have an endogenous<br />
control.<br />
Typically, housekeeping genes such as β-actin, glyceraldehyde-3-phosphate<br />
(GAPDH), and ribosomal RNA (rRNA), are used as endogenous controls.<br />
• Replicate wells – For relative quantification studies, Applied Biosystems<br />
recommends the use of three or more replicate reactions per sample and<br />
endogenous control to ensure statistical significance. Replicates allow you to<br />
preserve data and remove outliers.<br />
2<br />
For more information about these concepts, refer to the <strong>Sequence</strong> <strong>Detection</strong> <strong>Systems</strong><br />
Chemistry Guide.<br />
DRAFT<br />
September 26, 2003 3:14 pm, C2_Design.fm<br />
Notes<br />
<strong>Relative</strong> Quantification Getting Started Guide for <strong>7000</strong> v1.1 9