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原位点杂交：完整细胞中mRNA的定量分析

In situ dot blots: quantitation of mRNA in intact cells.

作者信息

Yu S M, Gorovsky M A

出版信息

Nucleic Acids Res. 1986 Oct 10;14(19):7597-615. doi: 10.1093/nar/14.19.7597.

DOI:10.1093/nar/14.19.7597
PMID:3095789
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC311783/
Abstract

A rapid, simple and reproducible dot blot method is described for quantitating the amounts of specific messages in small numbers of intact cells. The method has been used to accurately determine the number of histone H4 mRNA molecules in growing (approximately 40,000) and in starved (approximately 1600) Tetrahymena thermophila, and to measure the amount of message contributed by an E. coli plasmid containing part of the S10 ribosomal operon. Use of the method is illustrated to optimize in situ hybridization protocols and to measure mRNA amounts in cell lysates. Preliminary studies also indicate that the method can be used to detect mRNA in intact yeast cells.

摘要

本文描述了一种快速、简单且可重复的斑点印迹法，用于定量少量完整细胞中特定信使核糖核酸（mRNA）的含量。该方法已被用于准确测定生长状态（约40,000个）和饥饿状态（约1,600个）的嗜热四膜虫中组蛋白H4 mRNA分子的数量，并测量含有部分S10核糖体操纵子的大肠杆菌质粒所贡献的mRNA量。文中举例说明了该方法在优化原位杂交方案以及测量细胞裂解物中mRNA含量方面的应用。初步研究还表明，该方法可用于检测完整酵母细胞中的mRNA。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0918/311783/b829da128075/nar00288-0109-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0918/311783/e802876f2050/nar00288-0099-a.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0918/311783/891fd053d6a9/nar00288-0107-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0918/311783/89835f6298f4/nar00288-0109-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0918/311783/b829da128075/nar00288-0109-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0918/311783/e802876f2050/nar00288-0099-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0918/311783/f2c15d9422df/nar00288-0100-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0918/311783/ea6107a2d65a/nar00288-0106-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0918/311783/891fd053d6a9/nar00288-0107-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0918/311783/89835f6298f4/nar00288-0109-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0918/311783/b829da128075/nar00288-0109-b.jpg

相似文献

1
In situ dot blots: quantitation of mRNA in intact cells.原位点杂交：完整细胞中mRNA的定量分析
Nucleic Acids Res. 1986 Oct 10;14(19):7597-615. doi: 10.1093/nar/14.19.7597.
2
Multiple, independently regulated, polyadenylated messages for histone H3 and H4 in Tetrahymena.四膜虫中组蛋白H3和H4的多个独立调控的多聚腺苷酸化信使RNA
Nucleic Acids Res. 1983 Jun 25;11(12):3903-17. doi: 10.1093/nar/11.12.3903.
3
Unusual features of transcribed and translated regions of the histone H4 gene family of Tetrahymena thermophila.嗜热四膜虫组蛋白H4基因家族转录和翻译区域的异常特征。
Nucleic Acids Res. 1987 Jan 12;15(1):141-60. doi: 10.1093/nar/15.1.141.
4
Regulation of protein synthesis in Tetrahymena. RNA sequence sets of growing and starved cells.四膜虫中蛋白质合成的调控。生长细胞和饥饿细胞的RNA序列集。
J Biol Chem. 1983 Jun 10;258(11):6899-905.
5
Tetrahymena H4 genes: structure, evolution and organization in macro- and micronuclei.嗜热四膜虫H4基因：在大核和小核中的结构、进化及组织形式
Nucleic Acids Res. 1984 Feb 24;12(4):1961-75. doi: 10.1093/nar/12.4.1961.
6
Characterization of two types of histone H2B genes from macronuclei of Tetrahymena thermophila.嗜热四膜虫大核中两种组蛋白H2B基因的特性分析
Nucleic Acids Res. 1987 Jul 24;15(14):5681-97. doi: 10.1093/nar/15.14.5681.
7
Sequence and properties of the message encoding Tetrahymena hv1, a highly evolutionarily conserved histone H2A variant that is associated with active genes.编码嗜热四膜虫hv1的信息序列及特性，hv1是一种与活跃基因相关的高度进化保守的组蛋白H2A变体。
Nucleic Acids Res. 1988 Jan 11;16(1):179-98. doi: 10.1093/nar/16.1.179.
8
Regulation of protein synthesis in Tetrahymena. Quantitative estimates of the parameters determining the rates of protein synthesis in growing, starved, and starved-deciliated cells.四膜虫中蛋白质合成的调控。对生长、饥饿和饥饿去纤毛细胞中决定蛋白质合成速率的参数的定量估计。
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9
Intracellular distribution of histone mRNAs in human fibroblasts studied by in situ hybridization.通过原位杂交研究人成纤维细胞中组蛋白mRNA的细胞内分布。
Proc Natl Acad Sci U S A. 1988 Jan;85(2):463-7. doi: 10.1073/pnas.85.2.463.
10
Sensitive detection of RNAs in single cells by flow cytometry.通过流式细胞术对单细胞中的RNA进行灵敏检测。
Nucleic Acids Res. 1992 Jan 11;20(1):83-8. doi: 10.1093/nar/20.1.83.

引用本文的文献

1
Cloning and characterization of the major histone H2A genes completes the cloning and sequencing of known histone genes of Tetrahymena thermophila.嗜热四膜虫主要组蛋白H2A基因的克隆与特性分析完成了嗜热四膜虫已知组蛋白基因的克隆与测序。
Nucleic Acids Res. 1996 Aug 1;24(15):3023-30. doi: 10.1093/nar/24.15.3023.
2
Mapping the 5' and 3' ends of Tetrahymena thermophila mRNAs using RNA ligase mediated amplification of cDNA ends (RLM-RACE).利用RNA连接酶介导的cDNA末端扩增（RLM-RACE）技术对嗜热四膜虫mRNA的5'和3'末端进行定位。
Nucleic Acids Res. 1993 Oct 25;21(21):4954-60. doi: 10.1093/nar/21.21.4954.
3
Sequence and properties of the message encoding Tetrahymena hv1, a highly evolutionarily conserved histone H2A variant that is associated with active genes.

本文引用的文献

1
Regulation of protein synthesis in Tetrahymena: isolation and characterization of polysomes by gel filtration and precipitation at pH 5.3.嗜热四膜虫中蛋白质合成的调控：通过凝胶过滤和在pH 5.3下沉淀分离和鉴定多核糖体
Nucleic Acids Res. 1982 Mar 25;10(6):2145-61. doi: 10.1093/nar/10.6.2145.
2
Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with a biotinated nucleotide analog.通过使用与生物素化核苷酸类似物的原位杂交技术，在鸡肌肉组织培养物中观察到肌动蛋白基因的表达。
Proc Natl Acad Sci U S A. 1982 Dec;79(23):7331-5. doi: 10.1073/pnas.79.23.7331.
3
Regulation of protein synthesis in Tetrahymena. Quantitative estimates of the parameters determining the rates of protein synthesis in growing, starved, and starved-deciliated cells.
编码嗜热四膜虫hv1的信息序列及特性，hv1是一种与活跃基因相关的高度进化保守的组蛋白H2A变体。
Nucleic Acids Res. 1988 Jan 11;16(1):179-98. doi: 10.1093/nar/16.1.179.
4
A conditional mutant having paralyzed cilia and a block in cytokinesis is rescued by cytoplasmic exchange in Tetrahymena thermophila.一种具有麻痹纤毛和胞质分裂阻滞的条件突变体通过嗜热四膜虫的细胞质交换得以挽救。
Genetics. 1988 Nov;120(3):697-705. doi: 10.1093/genetics/120.3.697.
5
Localization and expression of mRNA for a macronuclear-specific histone H2A variant (hv1) during the cell cycle and conjugation of Tetrahymena thermophila.嗜热四膜虫细胞周期和接合过程中，大核特异性组蛋白H2A变体（hv1）mRNA的定位与表达
Mol Cell Biol. 1988 Nov;8(11):4780-6. doi: 10.1128/mcb.8.11.4780-4786.1988.
6
Direct measurement of tubulin and bulk message distributions on polysomes of growing, starved and deciliated Tetrahymena using RNA gel blots of sucrose gradients containing acrylamide.使用含有丙烯酰胺的蔗糖梯度的RNA凝胶印迹法，直接测量生长、饥饿和去纤毛的四膜虫多聚核糖体上微管蛋白和大量信息的分布。
Nucleic Acids Res. 1988 Oct 25;16(20):9597-609. doi: 10.1093/nar/16.20.9597.
7
A fast and sensitive method for detecting specific viral RNA in mammalian cells.
J Virol. 1988 Apr;62(4):1132-5. doi: 10.1128/JVI.62.4.1132-1135.1988.
8
Phylogenetic group-specific oligodeoxynucleotide probes for identification of single microbial cells.用于鉴定单个微生物细胞的系统发育组特异性寡脱氧核苷酸探针。
J Bacteriol. 1988 Feb;170(2):720-6. doi: 10.1128/jb.170.2.720-726.1988.
9
Macronuclei and micronuclei in Tetrahymena thermophila contain high-mobility-group-like chromosomal proteins containing a highly conserved eleven-amino-acid putative DNA-binding sequence.嗜热四膜虫的大核和小核含有类似高迁移率族的染色体蛋白，这些蛋白包含一个高度保守的十一个氨基酸的假定DNA结合序列。
Mol Cell Biol. 1991 Jan;11(1):166-74. doi: 10.1128/mcb.11.1.166-174.1991.
10
Replication-dependent and independent regulation of HMG expression during the cell cycle and conjugation in Tetrahymena.嗜热四膜虫细胞周期和接合过程中HMG表达的复制依赖性和非依赖性调控
Nucleic Acids Res. 1992 Dec 25;20(24):6525-33. doi: 10.1093/nar/20.24.6525.
四膜虫中蛋白质合成的调控。对生长、饥饿和饥饿去纤毛细胞中决定蛋白质合成速率的参数的定量估计。
J Biol Chem. 1983 Jun 10;258(11):6887-98.
4
Genome organization and reorganization in Tetrahymena.四膜虫的基因组组织与重组
Annu Rev Genet. 1980;14:203-39. doi: 10.1146/annurev.ge.14.120180.001223.
5
Detection of mrnas in sea urchin embryos by in situ hybridization using asymmetric RNA probes.使用不对称RNA探针通过原位杂交检测海胆胚胎中的mRNA。
Dev Biol. 1984 Feb;101(2):485-502. doi: 10.1016/0012-1606(84)90162-3.
6
Most early-variant histone mRNA is contained in the pronucleus of sea urchin eggs.大多数早期变体组蛋白mRNA包含在海胆卵的原核中。
Dev Biol. 1983 Nov;100(1):197-206. doi: 10.1016/0012-1606(83)90211-7.
7
Transcription of the S10 ribosomal protein operon is regulated by an attenuator in the leader.S10核糖体蛋白操纵子的转录受前导序列中一个衰减子的调控。
Cell. 1983 May;33(1):241-8. doi: 10.1016/0092-8674(83)90353-7.
8
In situ hybridization to study the origin and fate of identified neurons.原位杂交技术用于研究特定神经元的起源和命运。
Science. 1983 Nov 18;222(4625):800-8. doi: 10.1126/science.6356362.
9
Location of specific messenger RNAs in Caenorhabditis elegans by cytological hybridization.
Dev Biol. 1983 Jun;97(2):375-90. doi: 10.1016/0012-1606(83)90094-5.
10
Tetrahymena H4 genes: structure, evolution and organization in macro- and micronuclei.嗜热四膜虫H4基因：在大核和小核中的结构、进化及组织形式
Nucleic Acids Res. 1984 Feb 24;12(4):1961-75. doi: 10.1093/nar/12.4.1961.