Tips for microRNA quantification, over-expression, inhibition & more
For miRNA over-expression a good starting point to consider is mirVana miRNA Mimics available from Thermo Fisher Scientific. Make sure to also purchase a mirVana miRNA Mimic negative control. If you like you can also have a customer-defined mirVana miRNA Mimic synthesised to use as a positive control. This may be achieved by modifying the miRNA sequence to create an even stronger base pair match for your target gene of interest. Another option to consider to meet your miRNA over-expression needs is Exiqon, a company dedicated to solutions for RNA analysis.
For transfecting small products such as miRNA mimics or miRNA inhibitors a transfection reagent like Lipofectamine RNAiMAX from Thermo Fisher Scientific is a good option.
Assess protein changes
When assessing gene expression following miRNA over-expression or miRNA inhibition, besides assessing mRNA expression, it may also be wise to assess protein expression. While miRNA binding often leads to target mRNA degradation, in some cases the binding of miRNA does not result in mRNA degradation but rather effects gene expression via preventing the mRNA from being translated. In this event significant changes in protein expression may be observed whilst mRNA expression remains relatively unchanged.
miRNA quantification options
For miRNA quantification analysis qPCR options include SYBR green methods such as miScript assays available from Qiagen, and probe / fluorescent reporter methods such as Taqman microRNA assays available from Thermo Fisher Scientific. (For miScript assays Qiagen provides a miScript II RT kit to generate cDNA. For Taqman microRNA assays Thermo Fisher Scientific provides a Taqman microRNA reverse transcription kit to generate cDNA).
Endogenous normalisation controls for miRNA quantification
For miRNA quantification analysis RNU24 & RNU6B are two widely accepted options for endogenous quantitative normalisation controls.
miRNA target reporter vector assays
One of the best ways to confirm if a miRNA targets a particular gene (mRNA) sequence is to perform a reporter vector assay. To do so you will need to co-transfect cells with a reporter vector containing the target sequence you are testing and with miRNA mimics or inhibitors to manipulate the level of your miRNA of interest. As the expression levels of your miRNA change, the output signal from the vector should also change if the miRNA indeed targets that sequence. One of the best options available is the pmirGLO Dual-Luciferase miRNA Target Expression Vector from Promega combined with the Dual luciferase reporter assay system also from Promega. Promega recommends using a perfect match as a positive control. This may be achieved by custom synthesis of a perfect match miRNA mimic, or via a vector insert that is a perfect match for your miRNA of interest.