In general, it is recommended to use between two and five validated stably expressed reference genes for normalization. A normalization strategy based on a single non-validated reference gene leads to erroneous normalization up to 3 and 6.4 fold in 25 and 10% of the cases, respectively, with sporadic cases showing error values above 20. Normalization using multiple validated reference genes results in much more accurate resultsĪll too often a single reference gene is used for normalization. Therefore I will refer to reference genes for the remaining part of this article. Īccording to the MIQE guidelines ( ), genes used for normalization should be referred to as reference genes, not as housekeeping genes. qbase+ greatly facilitates the process of validating reference / housekeeping genes and performing state-of-the-art normalization using the geometric mean of multiple validated reference / housekeeping genes.īefore walking you through the tips, I just want to mention we recently recorded a. The use of multiple stable reference or housekeeping genes is generally accepted as the method of choice for RT-qPCR data normalization ( - ). The remaining technical variation should then be further reduced or removed by using a proper normalization approach, enabling a better appreciation of the true biological variation. It is highly recommended to minimize the technical variation by using standard operating procedures throughout the entire qPCR workflow. Influencing parameters include the amount and quality of starting material, enzymatic efficiencies, and overall transcriptional activity. Different variables, inherent to the RT-qPCR workflow need to be controlled for in order to minimize the technical variation. A measured difference in RNA expression level between two samples is the result of both true biological as well as experimentally induced (technical) variation.