Previously, a moderately repetitive DNA sequence （RRD3） was cloned from rice （Oryza sativa L.） by DNA renaturation kinetics. Sequence analysis revealed several conserved promoter motifs, including four TATA-boxes ...Previously, a moderately repetitive DNA sequence （RRD3） was cloned from rice （Oryza sativa L.） by DNA renaturation kinetics. Sequence analysis revealed several conserved promoter motifs, including four TATA-boxes and a CAAT-box, and promoter activity was shown in Escherichia coli and mammalian expression systems. Here, we inserted the RRD3 fragment into the plant promoter-capture vector, pCAMBIA1391Z, and examined whether the RRD3 fragment has promoter activity in plants. Transgenic tobacco and rice calli both showed β-glucuronidase （GUS） activity, indicating that RRD3 can act as a promoter in both monocot and dicot plants. Based on the promoter characteristic of RRD3, we designed a plant universal binary vector, pCRiRRD3, which is suitable for performing researches on plant RNA interference. This vector has two multiple cloning sites to facilitate sense and antisense cloning of the target sequence, separated by an intron fragment of 200 bp. The efficiency of the vector for gene silencing was assayed by histochemical and quantitative fluorometric GUS assays in transgenic tobacco. These research results suggested that this plant RNAi vector pCRiRRD3 can effectively perform gene silencing researches on both monocot and dicot plants.展开更多
Pyropia haitanensis has prominent stress-resistance characteristics and is endemic to China. Studies into the stress responses in these algae could provide valuable information on the stress-response mechanisms in the...Pyropia haitanensis has prominent stress-resistance characteristics and is endemic to China. Studies into the stress responses in these algae could provide valuable information on the stress-response mechanisms in the intertidal Rhodophyta. Here, the effects of salinity and light intensity on the quantum yield of photosystem II in Py. haitanensis were investigated using pulse-amplitude-modulation fluorometry. Total RNA and genomic DNA of the samples under different stress conditions were isolated. By normalizing to the genomic DNA quantity, the RNA content in each sample was evaluated. The cDNA was synthesized and the expression levels of seven potential internal control genes were evaluated using qRT-PCR method. Then, we used geNorm, a common statistical algorithm, to analyze the qRT-PCR data of seven reference genes. Potential genes that may constantly be expressed under different conditions were selected, and these genes showed stable expression levels in samples under a salinity treatment, while tubulin, glyceraldehyde- 3-phosphate dehydrogenase and actin showed stability in samples stressed by strong light. Based on the results of the pulse amplitude-modulation fluorometry, an absolute quantification was performed to obtain gene copy numbers in certain stress-treated samples. The stably expressed genes as determined by the absolute quantification in certain samples conformed to the results of the geNorm screening. Based on the results of the software analysis and absolute quantification, we proposed that elongation factor 3 and 18S ribosomal RNA could be used as internal control genes when the Py. haitanensis blades were subjected to salinity stress, and that a-tubulin and 18S ribosomal RNA could be used as the internal control genes when the stress was from strong light. In general, our findings provide a convenient reference for the selection of internal control genes when designing experiments related to stress responses in Py. haitanensis.展开更多
AIM: To establish methods for quantitative polymerase chain reaction (PCR) for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of hepatocellular carcinoma (HCC). METHODS: Total R...AIM: To establish methods for quantitative polymerase chain reaction (PCR) for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of hepatocellular carcinoma (HCC). METHODS: Total RNA from paraffin-embedded sections was isolated from 68 paraffin-embedded samples of HCC. Samples came from 54 male and 14 female patients with a mean age of 66.8 ± 7.8 years. Quantitative PCR was performed. Immunohistochemistry and in situ hybridization for hepcidin were also performed. RESULTS: Quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections of HCC was performed successfully. The expression level of hepcidin mRNA in cancer tissues was significantly higher than that in non-cancer tissues. A method of in situ hybridization for hepcidin was established successfully, and this demonstrated that hepcidin mRNA was expressed in non-cancerous tissue but absent in cancerous tissue. CONCLUSION: We have established novel methods for quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of HCC.展开更多
基金This work was supported by the National Natural Science Foundation of China (No. 30370809).
文摘Previously, a moderately repetitive DNA sequence （RRD3） was cloned from rice （Oryza sativa L.） by DNA renaturation kinetics. Sequence analysis revealed several conserved promoter motifs, including four TATA-boxes and a CAAT-box, and promoter activity was shown in Escherichia coli and mammalian expression systems. Here, we inserted the RRD3 fragment into the plant promoter-capture vector, pCAMBIA1391Z, and examined whether the RRD3 fragment has promoter activity in plants. Transgenic tobacco and rice calli both showed β-glucuronidase （GUS） activity, indicating that RRD3 can act as a promoter in both monocot and dicot plants. Based on the promoter characteristic of RRD3, we designed a plant universal binary vector, pCRiRRD3, which is suitable for performing researches on plant RNA interference. This vector has two multiple cloning sites to facilitate sense and antisense cloning of the target sequence, separated by an intron fragment of 200 bp. The efficiency of the vector for gene silencing was assayed by histochemical and quantitative fluorometric GUS assays in transgenic tobacco. These research results suggested that this plant RNAi vector pCRiRRD3 can effectively perform gene silencing researches on both monocot and dicot plants.
基金Supported by the National Natural Science Foundation of China(Nos.41476140,41306151,41676157,41506172)the Strategic Leading Science and Technology Projects of Chinese Academy of Sciences(No.XDA11020404)+1 种基金the China Postdoctoral Science Foundation(No.2015M582153)the Science and Technology Plan of Jiangsu Province(No.BE2016330)
文摘Pyropia haitanensis has prominent stress-resistance characteristics and is endemic to China. Studies into the stress responses in these algae could provide valuable information on the stress-response mechanisms in the intertidal Rhodophyta. Here, the effects of salinity and light intensity on the quantum yield of photosystem II in Py. haitanensis were investigated using pulse-amplitude-modulation fluorometry. Total RNA and genomic DNA of the samples under different stress conditions were isolated. By normalizing to the genomic DNA quantity, the RNA content in each sample was evaluated. The cDNA was synthesized and the expression levels of seven potential internal control genes were evaluated using qRT-PCR method. Then, we used geNorm, a common statistical algorithm, to analyze the qRT-PCR data of seven reference genes. Potential genes that may constantly be expressed under different conditions were selected, and these genes showed stable expression levels in samples under a salinity treatment, while tubulin, glyceraldehyde- 3-phosphate dehydrogenase and actin showed stability in samples stressed by strong light. Based on the results of the pulse amplitude-modulation fluorometry, an absolute quantification was performed to obtain gene copy numbers in certain stress-treated samples. The stably expressed genes as determined by the absolute quantification in certain samples conformed to the results of the geNorm screening. Based on the results of the software analysis and absolute quantification, we proposed that elongation factor 3 and 18S ribosomal RNA could be used as internal control genes when the Py. haitanensis blades were subjected to salinity stress, and that a-tubulin and 18S ribosomal RNA could be used as the internal control genes when the stress was from strong light. In general, our findings provide a convenient reference for the selection of internal control genes when designing experiments related to stress responses in Py. haitanensis.
基金Supported by A research grant from the Biomarker Society
文摘AIM: To establish methods for quantitative polymerase chain reaction (PCR) for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of hepatocellular carcinoma (HCC). METHODS: Total RNA from paraffin-embedded sections was isolated from 68 paraffin-embedded samples of HCC. Samples came from 54 male and 14 female patients with a mean age of 66.8 ± 7.8 years. Quantitative PCR was performed. Immunohistochemistry and in situ hybridization for hepcidin were also performed. RESULTS: Quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections of HCC was performed successfully. The expression level of hepcidin mRNA in cancer tissues was significantly higher than that in non-cancer tissues. A method of in situ hybridization for hepcidin was established successfully, and this demonstrated that hepcidin mRNA was expressed in non-cancerous tissue but absent in cancerous tissue. CONCLUSION: We have established novel methods for quantitative PCR for hepcidin using RNAs isolated from paraffin-embedded sections and in situ hybridization of HCC.
