FCRG - Fixation and Sorting for RNA.

Increasingly, Flow Cytometry Shared Resource Facilities are asked to sort cells for RNA isolation either in bulk or at the single cell level. In many cases, the ability to fix the cell prior to sorting is desirable. With so many fixation methods in the literature the Flow Cytometry Research Group (FCRG) decided to perform a systematic evaluation of the reported fixation methods to assess how the different fixatives affected the quality of RNA isolated from sorted cells. Based on the literature, five different common chemical fixatives were analyzed using the cell line HL-60. The assessment included a paraformaldehyde fixation, alcohol fixation (methanol and ethanol), formaldehyde fixation, zinc fixation and two commercial fixations, BDCytoperm/Cytofix(cat #554715) and eBiosciences Intracellular Fixation and Permeabilization Buffer (cat#88-8824-00). Each method was tested at two separate shared facilities and for each method different variations of the fixation procedureie, time, temperature, dilution were also tested. The protocol involved fixing the cells first then proceeding to sort those cells into lysis buffer (RLT) and measure the amount, quality, and purity of the RNA. Four samples were used for each fixation condition: unfixed not sorted, unfixed sorted, fixed not sorted, and fixed sorted. Nanodrop was used for purity, Ribogreenfor yield, and a bioanalyzer/qPCRfor quality. Results will be presented that will aid researchers and shared facilities in determining optimal fixation processes for experimental design involving cell sorting.