Design of a new plasma external reference and analysis of nucleic acid detection
Objective To design a novel plasma external reference (PLACON) to detect and analyze human plasma mRNA and miRNA. Methods A conserved sequence was selected from the genome of the low-level species Caenorhabditis elegans. It was designed as a novel plasma external reference and BLAST was used to dete...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | zho |
| Published: |
Editorial Office of Journal of Third Military Medical University
2019-07-01
|
| Series: | Di-san junyi daxue xuebao |
| Subjects: | |
| Online Access: | http://aammt.tmmu.edu.cn/Upload/rhtml/201812172.htm |
| _version_ | 1818402805196521472 |
|---|---|
| author | GU Jiaoyang NIU Kai YU Yongxin ZHENG Linpeng YANG Qiao |
| author_facet | GU Jiaoyang NIU Kai YU Yongxin ZHENG Linpeng YANG Qiao |
| author_sort | GU Jiaoyang |
| collection | DOAJ |
| description | Objective To design a novel plasma external reference (PLACON) to detect and analyze human plasma mRNA and miRNA. Methods A conserved sequence was selected from the genome of the low-level species Caenorhabditis elegans. It was designed as a novel plasma external reference and BLAST was used to detect the specificity of PLACON sequences. Plasma samples of patients with malignant tumors and cancer cell supernatants were collected. And PLACON, β-actin, GAPDH, and U6 which were added to the plasma or cell supernatant respectively, were used for amplification comparison to detect plasma mRNA, miRNA and cell supernatant mRNA, by quantitative real-time PCR (RT-qPCR). Results BLAST detected that the PLACON sequence had good specificity and no cross-correlation with the human genome. Melting curves showed high specificity in RT-qPCR reaction. The cycle threshold values of mRNA levels were significantly lower in plasma of PLACON group than the GAPDH and β-actin groups (P < 0.01). The cycle threshold (CT) value of miRNA level amplification in plasma in PLACON group was significantly lower than that in U6 group (P < 0.01). PLACON was validated in mRNA amplification experiments in cancer cell supernatants, and similar results were obtained. Conclusion PLACON sequence is specific, and shows better amplification advantage as internal reference in plasma, with high stability and efficient amplification. It can be used for quantitative detection of plasma mRNA and miRNA. |
| first_indexed | 2024-12-14T08:14:12Z |
| format | Article |
| id | doaj.art-5b3e2360157940fd86638ba5e01eac7a |
| institution | Directory Open Access Journal |
| issn | 1000-5404 |
| language | zho |
| last_indexed | 2024-12-14T08:14:12Z |
| publishDate | 2019-07-01 |
| publisher | Editorial Office of Journal of Third Military Medical University |
| record_format | Article |
| series | Di-san junyi daxue xuebao |
| spelling | doaj.art-5b3e2360157940fd86638ba5e01eac7a2022-12-21T23:09:59ZzhoEditorial Office of Journal of Third Military Medical UniversityDi-san junyi daxue xuebao1000-54042019-07-0141131239124510.16016/j.1000-5404.201812172Design of a new plasma external reference and analysis of nucleic acid detectionGU Jiaoyang0NIU Kai1YU Yongxin2ZHENG Linpeng3YANG Qiao4Cancer Institute of PLA, Second Affiliated Hospital, Army Medical University (Third Military Medical University), Chongqing, 400037, ChinaCancer Institute of PLA, Second Affiliated Hospital, Army Medical University (Third Military Medical University), Chongqing, 400037, ChinaCancer Institute of PLA, Second Affiliated Hospital, Army Medical University (Third Military Medical University), Chongqing, 400037, ChinaCancer Institute of PLA, Second Affiliated Hospital, Army Medical University (Third Military Medical University), Chongqing, 400037, ChinaCancer Institute of PLA, Second Affiliated Hospital, Army Medical University (Third Military Medical University), Chongqing, 400037, ChinaObjective To design a novel plasma external reference (PLACON) to detect and analyze human plasma mRNA and miRNA. Methods A conserved sequence was selected from the genome of the low-level species Caenorhabditis elegans. It was designed as a novel plasma external reference and BLAST was used to detect the specificity of PLACON sequences. Plasma samples of patients with malignant tumors and cancer cell supernatants were collected. And PLACON, β-actin, GAPDH, and U6 which were added to the plasma or cell supernatant respectively, were used for amplification comparison to detect plasma mRNA, miRNA and cell supernatant mRNA, by quantitative real-time PCR (RT-qPCR). Results BLAST detected that the PLACON sequence had good specificity and no cross-correlation with the human genome. Melting curves showed high specificity in RT-qPCR reaction. The cycle threshold values of mRNA levels were significantly lower in plasma of PLACON group than the GAPDH and β-actin groups (P < 0.01). The cycle threshold (CT) value of miRNA level amplification in plasma in PLACON group was significantly lower than that in U6 group (P < 0.01). PLACON was validated in mRNA amplification experiments in cancer cell supernatants, and similar results were obtained. Conclusion PLACON sequence is specific, and shows better amplification advantage as internal reference in plasma, with high stability and efficient amplification. It can be used for quantitative detection of plasma mRNA and miRNA.http://aammt.tmmu.edu.cn/Upload/rhtml/201812172.htmexternal referenceinternal referencert-qpcrmirna in plasma |
| spellingShingle | GU Jiaoyang NIU Kai YU Yongxin ZHENG Linpeng YANG Qiao Design of a new plasma external reference and analysis of nucleic acid detection Di-san junyi daxue xuebao external reference internal reference rt-qpcr mirna in plasma |
| title | Design of a new plasma external reference and analysis of nucleic acid detection |
| title_full | Design of a new plasma external reference and analysis of nucleic acid detection |
| title_fullStr | Design of a new plasma external reference and analysis of nucleic acid detection |
| title_full_unstemmed | Design of a new plasma external reference and analysis of nucleic acid detection |
| title_short | Design of a new plasma external reference and analysis of nucleic acid detection |
| title_sort | design of a new plasma external reference and analysis of nucleic acid detection |
| topic | external reference internal reference rt-qpcr mirna in plasma |
| url | http://aammt.tmmu.edu.cn/Upload/rhtml/201812172.htm |
| work_keys_str_mv | AT gujiaoyang designofanewplasmaexternalreferenceandanalysisofnucleicaciddetection AT niukai designofanewplasmaexternalreferenceandanalysisofnucleicaciddetection AT yuyongxin designofanewplasmaexternalreferenceandanalysisofnucleicaciddetection AT zhenglinpeng designofanewplasmaexternalreferenceandanalysisofnucleicaciddetection AT yangqiao designofanewplasmaexternalreferenceandanalysisofnucleicaciddetection |