xpression was assessed using Applied Biosystems 7900HT System S

xpression was assessed using Applied Biosystems 7900HT System. Statistical analysis of miRNA array data There were 664 miRNAs profiled for each of the 40 sam ples, Ct values were obtained with the automatic baseline and manual Ct set to 0. 1 threshold. Some miRNAs were only minimally expressed, and were excluded from further analyses, specifically we excluded those for which 20% or more of the samples had a missing Ct or Ct 35. Lowess smoothing was used to normalize measures across individuals. Missing values were imputed using a K Nearest Neighbour approach as described by Tusher et al. Any particularly extreme values for each miRNA were shrunk in towards the center of the distribution so as to lessen their influence. For each comparison of two groups, two sample t tests were used to assess nominal significance.

The Westfall Young min P approach using 1000 permutations of group labels was used to obtain p values adjusted for multiple testing. Empirical Batimastat q values were also estimated using the permuted data. Heat Maps and Box plots based on the miRNA array data Normalized Ct values were adjusted by subtracting the Ct value from an arbitrary constant value of 40 so that a higher adjusted Ct value would correspond to a higher miRNA expression. The table of adjusted Ct values for the 20 significantly dysregulated miRNAs between PGRN and PGRN FTLD TDP patients was loaded in Cluster 3. 0. A heat map showing the miRNA expression profiles for all the samples was gen erated after median centering the adjusted Ct values for each miRNA. The normalized and adjusted Ct values were summarized across groups with boxplots.

Validation of miRNA candidates in frontal cortex and cerebellum The top 20 miRNA candidates identified in the miRNA array experiment were selected for validation by qRT PCR in the same set of 8 PGRN and 32 PGRN FTLD TDP patient samples. In brief, 50 uls of reverse transcrip tion primers for the 20 miRNAs plus RNU48 as an endogenous control were divided into 3 primer pools, lyophilized, and subse quently resuspended in water for each pool resulting in 5�� multiplex RT primer pool. Total RNA was reverse transcribed in a 20 ul reaction volume using the miRNA Reverse Transcription Kit and 1 ul of cDNA was used in the Taqman miRNA assays.

Where duplicate Ct values differed by more than 2, the more extreme one relative to the distribution of Ct values across all samples was deleted, otherwise the mean of the duplicates was used as the final Ct for a tran script. Delta Cts were calculated by subtracting the Ct of the endogenous control RNU48. Minus delta Cts were used as the final values for analysis and assumed to represent the log base 2 of scaled expression levels. Two sample t tests and corresponding 95% confidence inter vals were used to compare groups, and the differ ences between means and CIs were exponentiated to provide fold change estimates under the assumption of perfect probe efficiency. For a total of 8 miRNAs validated in frontal cortex

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