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Michael H Farkas, Elizabeth D Au, Rosario Fernandez-Godino, Tadeusz J Kaczynski, Maria E Sousa; Characterizing lincRNA expression during RPE development. Invest. Ophthalmol. Vis. Sci. 2017;58(8):569.
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Long intervening non-coding RNAs (lincRNAs) are increasingly being implicated in human disease, yet nothing is known about their expression or function in the human retina. Using RNA-Seq, we analyzed both human induced pluripotent stem cell-derived RPE (iPS-RPE) and age matched human fetal RPE to characterize lincRNA expression during development.
iPS were grown in mTeSR media on matrigel coated plates. Embryoid bodies (EBs) were formed by lifting iPS colonies using accutase and were transitioned to neural iduction medium. EBs were then transferred to laminin coated plates, and RPE differentiation begun by adding retinal differentiation medium. At 90 days, RPE were dissected from the plates, pelleted, and flash frozen. Fetal RPE was isolated from 16 week whole eyes. An eye cup was made, and vitreous and neural retina were removed using hyaluronidase. RPE was isolated using dispase, pelleted, and flash frozen. RNA was isolated and RNA-Seq libraries were made using Agilent’s SureSelect Strand Specific RNA Library Preparation Kit. Samples were sequenced to a depth of 50 million reads/sample on an Illumina HiSeq 2500. Data were aligned to human genome build 38 with STAR, and gene counts generated using a comprehensive transcriptome annotation database composed of all empirically-determined transcripts. Differential expression was performed using DESeq.
In total, 22,816 transcripts (coding and non-coding) were expressed in iPS, 21,708 in iPS-RPE, and 20,978 in fetal RPE. We identified 1958 lincRNAs expressed in iPS-RPE and 1,964 in fetal RPE with a concordance of 73%. As many as 675 putative novel genes were also discovered, a majority of which are likely to be lincRNAs. We compared the expression of lincRNAs in undifferentiated iPS cells to iPS-RPE. In the differentiated cells, we found that 321 lincRNAs were turned on and 228 were turned off. Additionally, 279 lincRNAs were significantly (p<0.01) upregulated (>2-fold), and 234 were significantly downregulated.
This is the first characterization of lincRNA expression in human retinal tissue. We have identified thousands of expressed lincRNAs in the RPE and are likely to be important for RPE development and function. Since so little is known about lincRNA expression in the human retina, these data serve as a guide to expand our studies of lincRNA structure and function; thus providing the baseline for studying their role in visual dystrophies.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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