酿酒酵母
RNA沉默
分离(微生物学)
生物
病毒学
色谱法
化学
分子生物学
微生物学
酵母
核糖核酸
RNA干扰
生物化学
基因
作者
Fernando Cardoso,Alexandre Elias,Inês Pereira,Isabel Maurício,Olga Matos
出处
期刊:MethodsX
[Elsevier]
日期:2023-12-01
卷期号:11: 102435-102435
标识
DOI:10.1016/j.mex.2023.102435
摘要
Accurate genomic sequencing demands high-quality double-stranded RNA (dsRNA). Existing methods for dsRNA extraction from yeast, fungi, and plants primarily rely on cellulose, suitable only for small volume extractions, or the time-consuming lithium chloride precipitation. To streamline the traditional phenol-chloroform-based dsRNA extraction method, the main challenge is the reduction of mitochondrial DNA (mtDNA) and Single Stranded RNA (ssRNA) to no detectable levels after gel electrophoresis. This challenge is successfully addressed through the modified approach described here, involving phenol extraction at low pH, followed by the addition of ammonium sulfate to the aqueous buffer. The dsRNA isolated using this novel method exhibits comparable quality to that obtained through cellulose purification, and it is readily amenable to RT-PCR. Moreover, a single batch of yeast cell RNA isolation requires only 2-3 h of hands-on time, thus simplifying and expediting the process significantly.•Buffers were redesigned from [32,33,35].•No DNASE, Ribonuclease A or beads were used during the purification.•Simple and inexpensive dsRNA extraction and purification method is described.
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