Abstract
The isolation of high quality RNA is a crucial technique in plant molecular biology. The quality of RNA determines the reliability of downstream process like real time PCR. In this paper, we reported a high quality RNA extraction protocol for a variety of plant species. Our protocol is time effective than traditional RNA extraction methods. The method takes only an hour to complete the procedure. Spectral measurement and electrophoresis were used to demonstrate RNA quality and quantity. The extracted RNA was further used for cDNA synthesis, expression analysis and copy number determination through Real Time PCR. The results indicate that RNA was of good quality and fit for real time PCR. This high throughput plant RNA extraction protocol can be used to isolate high quality RNA from diverse plants for real time PCR and other downstream applications.
Highlights
High quality RNA extraction is an important step for gene expression studies
The ratio 260/230 is expected to be around 2 - 2.2. If this value is appreciably lower, it is an indication that contaminants such as carbohydrates, EDTA, guanidine isothiocyanate, and phenol that absorb at 230 nm are present in the sample
Melt curve analysis was carried out. In this result showed single peak, which determine the specificity of the reaction (Figure 6). These results indicated that RNA isolated by our protocol was of high quality and suitable for real time PCR with no interference in PCR amplification
Summary
High quality RNA extraction is an important step for gene expression studies. RNA is used for protein synthesis, widely employed in studies of gene expression pattern in different plants. Types and quantity of RNA in plants depends upon expression of particular genes, which leads to a particular phenotype. Obtaining a sufficient quantity of pure RNA is more challenging for generation sequencing of transcriptomes and Quantitative Real Time PCR (qPCR) analysis [1]. RNA is mostly single stranded, often contains ribose sugar that carries 2’ hydroxyl group that makes the RNA more subjected to hydrolysis than genomic DNA. It is more complicated to obtain high quality RNA especially when plant samples contain high level of RNAase, large quantities of polysaccharides, low concentration of nucleic acid (high water content), different types of
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