Our initial transcriptional analysis of lineage biased HSCs transcripts assayed 3 replicates of young, and 6 of each mid and old age populations. Correlation analysis of the HSCs purified from each age group showed the most variation between replicates in the mid-aged population and strongest correlation associated with age group and not HSC subtype. We cross compared the significantly up and down regulated genes in these populations, with previous generated Affymetrix expression data (MOE 430 2.0 arrays) of young and aged subsets of HSCs. Using a permissive cutoff for significance (p<0.05, FC 1.5) we compared differences between the subsets at each age (ySH vs ySL, mSH vs MSL, and oSH vs oSL) and also each compartment during aging (ySH vs oSH, mSL vs oSL, etc). Though CD150 is one of the few genes found in the overlap between young and aged transcripts that are significantly upregulated in the SH HSCs, there were very few total genes with consistent changes on both platforms. Age-related changes showed similarly poor overlap between comparisons of data generated from different platforms, with no discernable trends to explain the variation. We are currently examining the differentially expressed transcripts from the RNA sequencing data for pathway enrichment and will work towards validating expression differences found from both genomic platforms using qPCR. We have performed analysis on circRNA, ncRNAs formed from back-splicing events that largely serve as sequestering sponges for other classes RNA, and found significant expression of circRNA in all samples, with a total of 29,986 unique transcripts expressed in HSCs. More significant changes of circRNA were associated with age than with CD150 subtype, similar to the data for total transcripts. Further the mid-aged group is the most variable, and may require additional replicates. ESC is working to validate the expression of the circRNA and correlate their expression differences with total transcript level to establish the role of these non-coding RNA in the dysregulation of HSC potential.
