Journal: Scientific Reports

Article Title: Transcriptome analysis of developing lens reveals abundance of novel transcripts and extensive splicing alterations

doi: 10.1038/s41598-017-10615-4

Figure Lengend Snippet: Completely novel transcripts (CNTs) with high conservation score (phastCons Score > 0.8), and expressed in atleast one developmental stage are shown across the panels. Expression profiles are normalized by the maximum expression level of a given transcript across stages and hierarchically clustered using Cluster 3.0 and visualized as a heatmap using Java Treeview. Samples from E15.5 that came from a different study than the rest of the samples were excluded from this expression analysis in order to avoid the batch effect. Heat maps showing the expression profiles of ( a ) 647 completely novel (novelty score ≥ 70%) transcripts hierarchically clustered with representative transcript groups expressed ( b ) in only one specific developmental stage and ( c ) in all the developmental stages. Novelty score (NS) and phastCons score (PS) indices for transcripts are also shown in as an additional scale bar in each heat map. ( d ) RT-PCR analysis validates expression of two CNTs with a predicted ORF ( MSTRG.8249.1 and MSTRG.18685.1 ) and two CNTs with no known ORF ( MSTRG.17446.1 and MSTRG.21639.1 ) in E15.5, P0 and P10 lenses. Note that MSTRG.17446.1 is undetected in this analysis at stage E15.5. Hprt represents a loading control. Negative control is included for all CNTs tested where the RT-PCR reaction was performed using the same primers as for the CNTs but without any cDNA. Full-length gels are included in Supplementary Information file.

Article Snippet: RNA was treated with RNase free DNase (Qiagen Inc #79254, Valencia, CA). cDNA was synthesized from 200 ng of total RNA, representing three biological replicates at each developmental stage using Bio-Rad iScript TM cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA), and was used as a template in PCR analysis.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Negative Control

Journal: Scientific Reports

Article Title: Transcriptome analysis of developing lens reveals abundance of novel transcripts and extensive splicing alterations

doi: 10.1038/s41598-017-10615-4

Figure Lengend Snippet: Functional analysis and validation of the high confident exon skipping events discovered across lens developmental states. ( a ) Functional enrichment analysis of genes associated with high confidence (FDR 1%) skipped exon events identified using rMATS pipeline in atleast one pairwise comparison of developmental stages. For each biological process per group (color coded), the % genes per GO term with number of query genes (** in red) in the analysis is shown in histogram. This shows the functional grouping of the GO-terms based on GO hierarchy using the Cytoscape -ClueGO plugin. Significant clusters (p < 1e-2), color coded by group based on enriched GO-biological processes generated from ClueGO analysis with size of the nodes indicating level of significant association of genes per GO-term. ( b ) Experimental validation by RT-PCR analysis of a selected set of high confident skipped exonic events reveals that selected mRNA isoforms with skipped events are more abundant during embryonic and perinatal stages. The schematic of the expected products are shown next to the gene. For validation, primers (arrows) were designed on the exons (black box) flanking the alternatively spliced exon (grey box). For all the genes, band with higher molecular weight is the isoform including the alternatively spliced exon and band with lower molecular weight is the isoform with the skipped exon. Hprt represents a loading control. Negative control is included for all isoforms tested where the RT-PCR reaction was performed using the same primers as for the isoforms but without any cDNA. Full-length gels are included in Fig. .

Article Snippet: RNA was treated with RNase free DNase (Qiagen Inc #79254, Valencia, CA). cDNA was synthesized from 200 ng of total RNA, representing three biological replicates at each developmental stage using Bio-Rad iScript TM cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA), and was used as a template in PCR analysis.

Techniques: Functional Assay, Biomarker Discovery, Comparison, Generated, Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Control, Negative Control