PMC:4307275 / 15019-17664
Annnotations
{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/4307275","sourcedb":"PMC","sourceid":"4307275","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4307275","text":"2.2.2. Non-FCA-Based Analyses\nThe sGFP expression level and transfection ratio were analyzed using a non-FCA-based analysis (see Figure 4). The sGFP expressions were measured on molecular levels; mRNA transcripts by quantitative real-time PCR (see Figure 4A) and proteins by western blot analysis (see Figure 4B). As a result, both transcripts and proteins of K-sGFP FP did not considerably differ from those of the KR-sGFP. The KR-sGFP formed two protein bands of a premature-polypeptide of 32 kDa and a mature-polypeptide of 27 kDa produced after the cleavage of rRTp-transit peptide. This smaller one from KR-sGFP was supposed to be a functional form as a fluorophore emitter (see Figure 4B). The transfection ratio analysis using the hemocytometer showed no difference between K-sGFP and KR-sGFP (see Figure 4C). These results suggest that the localization of sGFP into chloroplasts does not affect the expression level of sGFP or transfection efficiency and support the presumption that the large population of the K-sGFP transfected protoplasts was analyzed as the non-transfected population with background signals because their fluorescence intensity was lower than the threshold value on FCA. The population with the lower fluorescence intensity would henceforth be called “the missing population” (M; see Figure 2C for its interval). This could also be supported by the images of hemocytometer analysis in the right panel of Figure 2C, on which fluorescence intensity and transfection ratio are not much different between K-sGFP and KR-sGFP.\nFigure 4 Comparison of K-sGFP and KR-sGFP constructs based on non-FCA analysis. (A) The mRNA transcripts of sGFP and rRTp-sGFP were compared using real-time PCR. The rice ubiquitin gene was used as an internal reference for quantitative normalization; (B) The expression efficiency of proteins compared by western blot analysis. Polyclonal rabbit antibody against sGFP was used at a titer of 1:5000 and the image of PAGE gel was used to show relative quantities of the loaded proteins. The graph shows the relative band intensities quantified using LAS4000 software and (C) The ratio of protoplast cells expressing sGFP proteins was analyzed by hemocytometer measurements. A volume of 10 μL of the transformed protoplasts was mounted on a hemocytometer and the images were obtained using the DIC and FITC-A channels of a confocal microscope (200× magnification). The sGFP-expressing cells were identified as the green fluorescent signals. The error bars show the SEM of data acquired from three independent transfections. Control samples were prepared by PEG transfection with no plasmid DNA.","divisions":[{"label":"title","span":{"begin":0,"end":29}},{"label":"p","span":{"begin":30,"end":1551}},{"label":"label","span":{"begin":1552,"end":1560}}],"tracks":[]}