Abstract
Purpose: :
miRNAs are small, non-coding, regulatory RNAs of 18-24 nucleotides in length found in all metazoans and provide a newly recognized level of regulation of gene expression. Previously, we compared miRNA expression profiles in adult mouse retina, brain and heart by microarray analysis and identified at least 78 miRNAs expressed in adult mouse retina, 15 of which are confirmed, by multiple tissue qRT-PCR, to be retina-specific or preferentially expressed in retina and brain. However, qRT-PCR analysis in retinas of different developmental stages revealed that most of these adult retinal miRNAs have low or no expression in embryonic stages, suggesting that different miRNAs may be expressed in various developmental stages in the retina ("developmental stage-specific miRNAs") to regulate the proliferation and sequential differentiation of the retinal progenitor cells. The purpose of the current study is to identify developmental stage-specific miRNAs,
Methods: :
Total RNA from eye cups of embryos at embryonic day (E)10, E14 and E18, as well as from retinas of postnatal days 1 (P1) and 10 (P10) mice was isolated using the mirVana miRNA Isolation system (Ambion). Small-sized RNA (<40 nt) from the total RNA samples using a FlashPAGE Fractionator (Ambion) according to the manufacturers’ protocol. We used the mirVana miRNA labeling Kit (Ambion) to label the miRNA with Cy3. mirVana miRNA Bioarray-v2 (Ambion) was employed for the miRNA profiling. The arrays were scanned at 90-100% power and a photomultiplier (PMT) of 75-80 and 5 µm resolution using Packard Biochip scanner and ScanArray software. The spot intensity was determined in QuantArray. The data analysis was performed as decribed previously (Xu et al. 2007).
Results: :
1) miRNA transcriptomes of the retina changes along retinal development; 2) the changes in the retinal transcriptomes reflect on both the composition of expressed miRNAs at each developmental stage, and the level of expression of each expressed miRNA; 3) each miRNA expressed during the retinal development follows a dynamic, time-sensitive pattern along the development; 4) various miRNAs peak at different developmental stages, constituting a "stage-specific" miRNA combination or "signatures".
Conclusions: :
Developmental stage-specific miRNAs may constitute a miRNA signature of retinal development. These stage-specific miRNAs may play key roles in the fine-tuning of the proliferation/differentiation of the retinal progenitor cells to contribute to the tightly controlled spatial and temporal sequences in retinal development. These miRNAs may become invaluable tools in sequential differentiation of retinal progenitor cells to mature retinal cells.
Keywords: retinal development • gene/expression • gene microarray