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Normalization Methods of miRNA Quantification - Ask TaqMan #40

What do Endogenous Controls, Exogenous Controls and Mean Expression value mean to you? You guessed it! Today we’re answering your questions about normalization methods for miRNA quantification. MicroRNAs or miRNAs, are small ~22 nucleotide noncoding RNAs that regulate gene expression. microRNAs can be quantified by real-time PCR using TaqMan assays. In a microRNA expression experiment, variation in the amount of starting material, sample collection, RNA preparation and quality, and reverse transcription efficiency can contribute to quantification errors. For these reasons, it is important to use proper normalization controls when quantifying miRNAs. There are three types of normalization methods commonly used for miRNA analysis by qPCR - endogenous controls, exogenous controls and mean expression value normalization, or “global mean normalization” Normalization using endogenous control genes is currently the most accurate method to correct for potential differences in RNA input or RT efficiency biases. Exogenous controls or “spike-ins” are typically used to monitor extraction efficiency or sample input amount for difficult samples such as plasma/serum or other biofluids. Large scale miRNA expression profiling studies may utilize global mean normalization, which uses the calculated mean of all miRNAs in a given sample as the normalizer. Historically, non-coding RNAs such as snRNAs and snoRNAs were used as endogenous normalizers for miRNA quantification. But, more recently, key opinion leaders in the miRNA community have moved away from the use of snoRNAs/snRNAs as endogenous controls for the following reasons: • They are bigger than miRNAs • They do not ‘mirror’ the physiochemical properties of miRNA • They have different cellular processing and different functions than miRNAs • The expression levels of snoRNA and snRNA have been recently found to be associated with cancer and prognosis. The miRNA community has also suggested that the ideal endogenous control has gene expression that is relatively constant and moderately abundant across a variety of tissues and cell types and treatment. miRNAs that are uniformly expressed can be used as an endogenous control. There are several miRNAs that have been shown in the literature and in experimental studies to be expressed at relatively constant levels across many different tissues types (show table). These may work as good endogenous controls for your experimental condition. By the way, it is recommended to validate 2 or more of these miRNAs as endogenous controls for the target cell, tissue, or treatment that you are using because no single control can act as a universal endogenous control for all experimental conditions. In addition, synthetic miRNA molecules can be used as spike-in controls and are extremely useful as exogenous controls in difficult samples such as serum/ plasma. As the name implies, spike- ins, or exogenous controls are synthetic RNA oligonucleotides that are added to the sample. A spike-in control should be a target sequence that is not present in your sample. For example, ath-miR-159a (need pronunciation guidence) is not present in humans so it is a good exogenous control for human. So let’s do a quick recap. There are 3 different normalization methods, exogenous controls, endogenous controls and mean expression value normalization and these normalization methods allow you to control certain aspects of your experimental process when analysing miRNAs by qPCR. If you have more questions on Normalization methods or any burning qPCR questions make sure to let me know and Ask TaqMan at http://www.thermofisher.com/ask