spike-in control - (Nov/08/2019 )

Hello,

Im new to microRNA. As all qPCR reactions have to be normalised, can someone help  me understand what is spike-in control for miRNA normalization and how it work?

When do you we use endogenous miRNA-xx and when do we use spike-in control for quantification, doe sit depend on sample type?

thank you

A spike-in control is when you add a known amount of a known target to a reaction. You use this when you need to check that your reactions are working as expected and your data is the result of correct amplification of your target genes. You wouldn't normally use it as a standard for quantitation other than you can use it to create a standard curve for copy-number determination.

Thank you.

But does addition of spike-in miRNA affect the RNA concentration and give overestimated readings?

1. Spike is added during RNA extraction of after elution of RNA?

You can use it where ever you need to test. If you want to test your extraction procedures, then spike it into a control sample before extraction, then see how much comes through in your assay. In this case it will not affect the RNA concentration. You can add it to the PCR at known concentration(s) to give you control amplification for copy-number analysis. In this case you wouldn't necessarily add it to a RNA extraction, just spike it straight into the PCR.

You can also spike it into a RNA extraction before PCR to test for presence of inhibitors in the RNA extraction itself.