The microRNA expression profile of mouse Muller glia in vivo and in vitro. Mus musculus

In order to identify the miRNAs in adult and young (postnatal day 11/12) Müller glia of the neural retina, we isolated the Müller glia from Rlbp-CreER: Stopf/f-tdTomato mice by means of fluorescent activated cell sorting and analyzed their miRNAs using NanoStrings Technologies®. We next compared miRNA expression of acutely isolated Müller glia with those that were maintained in dissociated culture for 8 and 14 days. We found that most miRNAs declined in vitro. Interestingly, some miRNAs that were not highly expressed in adult Müller glia increased in cultured cells. Our results thus show the miRNA profile of adult Müller glia and the effects of cell culture on their levels. Overall design: We used fluorescence activated cell sorting (FACS) to purify adult MG (pooled from 40 retinas), using an MG specific cre-lox labeling strategy by crossing Rlbp-CreER mice with stopf/f-tdTomato mice. The RNA was extracted from purified MG (tdTomato+) and retinal neurons (tdTomato−) and miRNA expression was analyzed by means of a recently developed molecular barcode technology called NanoStrings®. The miRNAs of freshly isolated P11/12 (pooled from 32 retinas) and adult Muller glia and cultured Muller glia (8 and 14 days in vitro, 20 retinas total) were analyzed using NanoString and nCounter Technologies ®. [1] Values from adult MG, adult neurons, P11 MG and P11 neurons were normalized against 4 housekeeping genes (β -actin, GAPDH, Rpl19, and B2m), 8 negative controls, and 6 positive controls that were run simultaneously with the samples. [2] Values from adult MG, P11 MG and cultured MG for 8 DIV or 14 DIV were normalized against 4 housekeeping genes (β -actin, GAPDH, Rpl19, and B2m), 8 negative controls, and 6 positive controls that were run simultaneously with the samples. This work was funded by these additional grants: Grant Number: Wo 2010/1-1 Source: DFG scholarship for S.G. Wohl, German Research Foundation (DFG) Grant Number: TA-RM-0614-0650 Source: for T.A. Reh, Foundation Fighting Blindness (FFB) Grant Number: P30EY01730 Source: Vision Core Grant for the use of imaging facilities