FAQ: Why is it important to evaluate RNA damage or inhibition during cell lysis and one-step RT-qPCR detection?

At high concentrations, certain cellular components may damage RNA or inhibit cell lysis and RT-qPCR steps, leading to inaccurate quantitation of RNA expression. In the presence of the Luna Cell Ready RNA Protection Reagent, RNA damage can be dramatically reduced. 

Two approaches can be used to evaluate proper performance of the Luna Cell Ready workflow. 

1) Spike in a reference RNA when preparing your cell lysis mix. (Compare the Cq values of your reference RNA in the presence or absence of cells. Similar Cq values (≤1 Cq difference) indicate limited RNA damage and minimal inhibition to RT-qPCR reactions. It is recommended to use a reference RNA without homology to the RNA population. For example, if you are working with human cell lines, in vitro transcribed Lambda phage RNA may be an option.)

2) Perform one-step RT-qPCR with purified total RNA approximately equivalent to the amount of RNA in the cell lysate. For example, when the cells are harvested, half of the amount can be used for RNA extraction using a column-based kit (e.g., Monarch Total RNA Miniprep Kit, NEB#T2010S) while the other half can be prepared with the Luna Cell Ready Lysis Module. 

Similar Cq values suggest efficient cell lysis. Otherwise, decrease the cell number until the similar results are achieved.