RNA recovery and detection of mRNA by RT-PCR from preserved prokaryotic samples
RNA recovery and detection of mRNA by RT-PCR from preserved prokaryotic samples
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DOI：
10.1111/j.1574-6968.2001.tb10745.x
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发表时间：
2001-07-24
影响因子：
2.1
通讯作者：
Hodson, RE
中科院分区：
文献类型：
--
作者：
Bachoon, DS;Chen, F;Hodson, RE
比较了在聚球藻和假单胞菌细胞中保存原核RNA，在96%乙醇、4%多聚甲醛中固定，或悬浮在RNAlater中，并冷藏3个月的有效性。从聚球藻和假单胞菌细胞提取的RNA的荧光测定表明，测试的细胞储存处理在保持其总RNA含量方面同样有效。在储存期间，从保存的样品中分离的RNA的量没有任何可检测的减少。通过逆转录酶-聚合酶链反应（RT-PCR）从储存3个月的保存细菌细胞中检测到RuBisCO（rbcL）和nir基因的完整mRNA转录本。相反，RT-PCR未能扩增rbcL和亚硝酸盐还原酶基因的mRNA在未固定和/或未保存的细菌样品，这表明细菌的mRNA可以很好地保持在一个长期的存储过程中，当细胞被妥善保存。此外，RNAlater是一种有用的试剂，用于储存和维持未冷冻样品中的高质量RNA。(C)2001年，欧洲微生物学会联合会。由Elsevier Science B. V.出版，版权所有。
The effectiveness of maintaining prokaryotic RNA in Synechococcus and Pseudomonas cells, fixed in 96% ethanol, 4% paraformaldehyde, or suspended in RNAlater, and held in cold storage for 3 months was compared. Fluorometric determination of the RNA extracted from Synechococcus and Pseudomonas cells indicated that the cell storage treatments tested were equally effective at maintaining their total RNA content. There was not any detectable decrease in the quantity of RNA isolated from the preserved samples during storage. Intact mRNA transcripts of the RuBisCO (rbcL) and nir genes were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) from preserved bacterial cells throughout 3 months of storage. In contrast, RT-PCR failed to amplify the mRNA of the rbcL and nitrite reductase genes in unfixed and/or unpreserved bacterial samples, suggesting that bacterial mRNA can be well maintained during a prolonged storage when cells are preserved properly. In addition, RNAlater is a useful reagent for the storage and maintenance of high quality RNA in unfrozen samples. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.