AMPLIFIED RNA SYNTHESIZED FROM LIMITED QUANTITIES OF HETEROGENEOUS CDNA
AMPLIFIED RNA SYNTHESIZED FROM LIMITED QUANTITIES OF HETEROGENEOUS CDNA
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DOI：
10.1073/pnas.87.5.1663
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发表时间：
1990-03-01
影响因子：
11.1
通讯作者：
EBERWINE, JH
中科院分区：
文献类型：
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作者：
VANGELDER, RN;VONZASTROW, ME;EBERWINE, JH
神经基因表达的异质性和大脑中许多低丰度信使 RNA 的空间有限表达使得此类信息的克隆和分析变得困难。为了产生足以用于标准克隆策略的核酸量，我们设计了一种从有限数量的 cDNA 产生扩增的异质 RNA 群体的方法。用含有 T7 RNA 聚合酶启动子序列 5' 至多胸苷酸区域的合成寡核苷酸引发整个小脑 RNA。第二链 cDNA 合成后，T7 RNA 聚合酶用于生成扩增的反义 RNA (aRNA)。纳克级 cDNA 的摩尔扩增高达 80 倍。扩增材料的大小分布与亲本 cDNA 相似，并通过 Southern 和 Northern 印迹分析评估显示出序列异质性。在扩增材料中已检测到肌动蛋白和鸟嘌呤核苷酸结合蛋白（G 蛋白）α 亚基的中等丰度 mRNA 的特定信息。通过使用原位转录生成 cDNA，在源自单个小脑组织切片的 aRNA 中检测到了亲环蛋白序列。源自单个小脑浦肯野细胞的 cDNA 也已被扩增，并产生与同源完整 RNA 和 mRNA 杂交但不与大肠杆菌 RNA 杂交的材料。
The heterogeneity of neural gene expression and the spatially limited expression of many low-abundance messenger RNAs in the brain has made cloning and analysis of such messages difficult. To generate amounts of nucleic acids sufficient for use in standard cloning strategies, we have devised a method for producing amplified heterogeneous populations of RNA from limited quantities of cDNA. Whole cerebellar RNA was primed with a synthetic oligonucleotide containing the T7 RNA polymerase promoter sequence 5' to a polythymidylate region. After second-strand cDNA synthesis, T7 RNA polymerase was used to generate amplified antisense RNA (aRNA). Up to 80-fold molar amplification has been achieved from nanogram quantities of cDNA. The amplified material is similar in size distribution to the parent cDNA and shows sequence heterogeneity as assessed by Southern and Northern blot analysis. Specific messages for moderate-abundance mRNAs for actin and guanine nucleotide-binding protein (G-protein) alpha subunits have been detected in the amplified material. By using in situ transcription to generate cDNA, sequences for cyclophilin have been detected in aRNA derived from single cerebellar tissue sections. cDNA derived from a single cerebellar Purkinje cell also has been amplified and yields material that hybridizes to cognate whole RNA and mRNA but not to Escherichia coli RNA.