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Tips for finding reliable housekeeping genes

Commonly used RT-qPCR housekeeping genes perform variably under different experimental conditions, so how can you reliably normalize RNA expression? Many researchers have uncovered outstanding new controls by mining relevant public datasets for genes that vary least across samples. Doing so has helped them identify both highly and moderately expressed genes.

If you don't have access to relevant datasets or the capability to mine them, you can create a good shortlist of potential housekeeping genes through literature searches. After identifying prospects, you can make a final choice by running an RT-qPCR assay with samples that highlight various conditions or subgroups in your experiment. The best control genes will always be those that demonstrate the greatest stability in your experimental system.