Application of laser-induced fluorescence for quantitative analysis of messenger RNA

激光诱导荧光在信使 RNA 定量分析中的应用

• 批准号: 07556115
• 负责人: SAKAI Senkiti
• 金额: $ 0.77万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07556115/
• 关键词: compctitive RT-PCR; fluorescene-labeled probe; capillarv clectrophoresis; casein; casein mRNA; fluorescene; mammary gland; competitor DNA; ノーザンブロット; RNA分析法; FITCラベル; ラベルプローブ; レーザー分析法

Cloning of casein mRNA and competitive reverse-transcription/polymerase chain reaction (RT-PCR) mRAN was extracted from lactating mouse mammary gland. Among casein mRNAs, mRNA for casein with the molecular weight of 22,000 was transcribed to cDNA and cloned. The nucleic acid sequence of casein cDNA was determined. DNA fragment of known foreign gene was inserted into casein cDNA.This kimeric DNA was used as competitor DNA during the polymerase chain reaction.Animals and assay conditions The quantity of casein mRNA in the mammary gland was determined by the RT-PCR throughout lactation. Its quantity was small at early lactation, reached maximum at midlactation, and declined gradually toward to the end of lactation. By the removal of pups from the midlactating mother, casein mRNA began to disappear from the mammary gland at 9h after the separation. By supplying the foser pups, casein mRNA increased and returned to the normal level.Fluorescene-labelled probe First, casein cDNA was amplified with fluorescene-labelled UTP by the RT-PCR.The incorporation efficiency was very poor. Secondly, the casein DNA fragment of 400 bp in lenght was inserted into the plasmid DNA.After the digestion of the DNA with Spe I,the fluorescene-labelled sense and antisense RNA probes were synthesized using T7 and SP6 polymerases, respectively. We detected the fluorescene-labelled RNA probe with the amount of larger than 10^<-15> mol and could be used for tne Northern blot analysis and for capillary clectrophoresis.

从泌乳小鼠乳腺中提取酪蛋白mRNA并进行竞争性逆转录/聚合酶链反应（RT-PCR）。在酪蛋白mRNA中，将分子量为22，000的酪蛋白的mRNA转录为cDNA并克隆。测定了酪蛋白cDNA的核酸序列。将已知外源基因的DNA片段插入到酪蛋白cDNA中，作为PCR反应的竞争DNA。动物和测定条件用RT-PCR方法测定泌乳期奶牛乳腺中酪蛋白mRNA的含量。其数量在泌乳早期较少，在泌乳中期达到最大值，并逐渐下降到泌乳结束。从哺乳中期的母亲，从9小时后的分离，酪蛋白mRNA开始从乳腺中消失的幼仔。用荧光素标记的UTP进行RT-PCR扩增酪蛋白cDNA，结果发现，酪蛋白cDNA的掺入效率很低，但在100 ℃时，掺入效率很低。然后将400 bp的酪蛋白DNA片段插入质粒DNA中，用Spe Ⅰ酶切后，分别用T7和SP 6聚合酶合成荧光标记的正义和反义RNA探针。荧光标记的RNA探针的量大于10 μ <-15>mol，可用于北方印迹分析和毛细管电泳。

项目成果

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Mizoguchi, Y., Kim, Y.J., Sakai, S.et al.：“早孕小鼠乳腺催乳素受体基因表达的调节”Endocrine J. 44 (1)。

Mizoguchi,Y.,Kim,J.Y.ら: "The regulation of the Prolactin Receptor Gene Expression in the Mammary Gland of Early Pregnent Mouse" Endocrine Journal. 44・1(印刷中). (1997)

Mizoguchi, Y., Kim, J.Y. 等：“早期妊娠小鼠乳腺中催乳素受体基因表达的调节”内分泌杂志 44·1（出版中）。

Mizoguchi,Y.,Kim,J.Y.J: "Acute Expression of the Prolactin Receptor Gene after Ovariectonamy in Midprequant Mouse Mammary Gland" Endocrine Journal. 43・5. 537-544 (1996)

Mizoguchi, Y., Kim, J.Y.J：“中期小鼠乳腺卵巢切除术后催乳素受体基因的急性表达”内分泌杂志 43・5 (1996)。

Hondo,E.,Kurohmaru,M.ら: "Prolactin Receptor Expression in Rat Spermatogenic Cells" Biology of Reproduction. 52巻. 1284-1290 (1995)

Hondo, E., Kurohmaru, M. 等人：“大鼠生精细胞中的催乳素受体表达”，《生殖生物学》，第 52 卷，1284-1290 (1995)。

Mizoguchi, Y., Kim, Y.J., Sakai, S., et al.: "Acute expression of the prolactin receptor gene after ovariectomy in midpregnant mouse mammary gland" Endocrine J.43 (5). 537-544 (1996)

Mizoguchi, Y., Kim, Y.J., Sakai, S., et al.：“中期妊娠小鼠乳腺卵巢切除术后催乳素受体基因的急性表达”内分泌 J.43 (5)。