Journal: PLoS ONE

Article Title: Cadherins in the retinal pigment epithelium (RPE) revisited: P-cadherin is the highly dominant cadherin expressed in human and mouse RPE in vivo

doi: 10.1371/journal.pone.0191279

Figure Lengend Snippet: (A) A method for extracting RNA individually from mouse RPE and choroid was established, and RNA samples were tested for cross-contamination. The expression of RPE markers ( Sox9 , Otx2 , and Rpe65 ) in three biological replicates was analyzed by RT-qPCR in triplicate using Gapdh , Hprt , and Actb as reference genes. Relative RNA quantity was calculated as a ratio to the expression level in mouse RPE samples. The values represent the means and SEM (bar). (B) The same RNA samples were tested for cross-contamination using choroid markers ( Vwf and Col6a1 ) by RT-qPCR in the same manner as in A. Relative RNA quantity was calculated as a ratio to the expression level in mouse choroid samples. The values represent the means and SEM (bar). (C) Total RNA from mouse RPE and choroid was prepared individually using the newly established method, and the mRNA expression of three cadherins was tested. RT-qPCR analysis was performed for Cdh1 (gene for E-cadherin), Cdh2 (N-cadherin), and Cdh3 (P-cadherin) in the same manner as described in A. Relative expression was calculated as a ratio to the expression level in mouse RPE. The values represent the means and SEM (bar). Statistical significance is shown by * (p < 0.05) and ** (p < 0.01).

Article Snippet: We designed all cadherin primers for PCR based on the known RefSeq mRNA sequence for a preproprotein that undergoes proteolytic processing to generate a mature protein (National Center for Biotechnology Information, NCBI).

Techniques: Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Cadherins in the retinal pigment epithelium (RPE) revisited: P-cadherin is the highly dominant cadherin expressed in human and mouse RPE in vivo

doi: 10.1371/journal.pone.0191279

Figure Lengend Snippet: (A) Absolute quantification of cDNA to assess the mRNA quantity of Cdh1 , Cdh2 , and Cdh3 in mouse RPE in situ . Total RNA was prepared from the RPE of 2 week-old and 2 month-old mice, and RT-qPCR was performed, along with gel-purified PCR products to create standard curves ranging from 1 attomole (amole) to 0.1 zeptomole (zmole). Based on Ct values of the standard curves, the quantity of cDNA for each gene was calculated for 200 ng total RNA used for cDNA synthesis. Three biological replicates were analyzed in triplicate for each sample. The values represent the means and SEM (bar). (B) Absolute quantification of cDNA to assess the mRNA quantity of CDH1 , CDH2 , and CDH3 in human RPE. Total RNA was prepared from the RPE of two donor eyes (RPE-1 and RPE-2) and human RPE primary cells (M1), and RT-qPCR was performed in triplicate in the same manner as described in A, along with gel-purified PCR products to create standard curves. Based on Ct values, the quantity of cDNA for each gene was calculated for 200 ng total RNA. The values represent the means and SEM (bar).

Article Snippet: We designed all cadherin primers for PCR based on the known RefSeq mRNA sequence for a preproprotein that undergoes proteolytic processing to generate a mature protein (National Center for Biotechnology Information, NCBI).

Techniques: Quantitative Proteomics, In Situ, Quantitative RT-PCR, Purification, cDNA Synthesis