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The importance of controls

Success with any assay begins with good and consistent quality control practices. ACD recommends two levels of quality control: a technical workflow check and a sample/RNA quality check.

Technical workflow quality control

A technical quality control check ensures that the assay is working correctly. This is easily performed using a control sample tested on two separate slides – one slide with a housekeeping gene positive control probe and another slide with a nonspecific bacterial gene negative control probe (i.e., dapB). When the assay is run successfully, the positive control slide will display strong staining and the negative control slide will display no staining. Including technical quality controls with every assay is recommended.

Sample/RNA quality control

Although the RNAscope® assay has universal conditions that work on most samples, tissue RNA quality and fixation conditions can vary. As a result, it is occasionally necessary to adjust the pretreatment conditions for optimal results. Section 1.3.1 of this book provides guidance on the optimization of pretreatment conditions, and section 2 details the optimal pretreatment conditions for 24 tissue types in rat, dog and cynomolgus monkey. However, checking tissue sample quality with positive and negative controls before running experiments is also recommended. This should result in a high positive control signal and no negative control background. If signal is low or background is detected, this is most often improved with adjustment to the pretreatment conditions as discussed in section 1.3.1.

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