頁籤選單縮合
題 名 | Role of the Exon 6 of Human Pdgf A-Chain mRNA in the Determination of Gene Expression by Reverse Transcription-Polymerase Chain Reaction=人類血小板生長因子A鏈mRNA之表現序列6對利用RT-PCR測定其基因表現之影響 |
---|---|
作 者 | 郭國華; | 書刊名 | The Kaohsiung Journal of Medical Sciences |
卷 期 | 13:2 1997.02[民86.02] |
頁 次 | 頁59-66 |
分類號 | 364.21 |
關鍵詞 | 血小板生長因子; A鏈mRNA; 序列6; RT-PCR測定; Alternative-spliced PDGF A; Exon 6; RNA secondary structure; |
語 文 | 英文(English) |
中文摘要 | 人類血小板生長因子A鏈Mrna係由不同接合作用即含 (PDGFA[+6]) 與不含 (PDGFA[-6]) 表現序列6之兩種Mrna所組成,為探討表現序列6對細胞內血小板生長因子A鏈Mrna基因表現之影響,我們設計出表現與插入序列夜接處之引子,分別以RT-PCR偵測其不同接合作用之Mrna表現。結果 (PDGFA[-6]) Mrna在黏合溫度60°C、65°C、70°C及75°C下均可被放大出;而PDGFA[+6] Mrna側僅可在70°C被作用,顯示PDGFA[+6] Mrna的二級結構可能影響RT-PCR偵測基因表現。同時PDGFA[+6]Mrna在反應上亦無法與PDGFA[-6]競爭共同引子。另一方面,人類血小板生長因子A鏈Mrna在含有表現序列6之核酸順序下醬形成高度互補與雙鏈RNA結構。根據研究報告Mrna的體外放大作用會受到其二級結構的影響,本篇PDGFA[+6]之RT-PCR情形與其RNA二級構造的結果是相符合的。因此綜合以上結果顯示,人類血小板生長因子A鏈Mrna之表現序列6的結構將影響RT-PCR對人類血小櫳生長因子A鏈Mrna基因表現之測定,同時雜合PDGFA[+6]Mrna所需之較嚴厲溫度條件,亦可能解釋利用一般方法並無法有效偵測細胞中PDGFA[+6]Mrna的原因。 |
英文摘要 | Alternative-spliced PDGF A mRNA is constituted with exon 6-excluded (PDGFA[-6]) and exon 6-included (PDGFA[+6]) transcripts. To define the role of exon 6 in the gene expression of alternative-spliced PDGF A in U937 cell, the exon-junction primers were employed for RT-PCR. The PDGFA[-6] mRNA could be amplified with specific primers at annealing temperatures of 60°C, 65°C, 70°C and 75°C, whereas PDGFA[+6] mRNA could be detected only at 70°C, suggesting that the RNA secondary structure of PDGFA[+6] might affect the RT-PCR reactions. In addition, PDGFA[+6] mRNA was incapable of competing with PDGFA[-6] mRNA for the common primers. A highly complementary and double-stranded RNA structure was formed for PDGF a when the exon 6 was included in the sequence. Since accessibility of a RNA template for in vitro amplification is related to RNA secondary structure, the results derived from RT-PCR and mRNA folding of PDGFA[+6] are essentially consistent. Thus, these results suggest that the exon 6 may affect the determination of gene expression by constricting the RNA secondary structure of PDGF A. The requirement of imperatively high stringency in the hybridization conditions for PDGFA[+6] mRNA may account for the low detection of the transcript in cells by conventional methods. |
本系統中英文摘要資訊取自各篇刊載內容。