Tropical Plant Research
An International Journal by Society for Tropical Plant Research
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2018, VOLUME 5 ISSUE 1Pages: 08-13
Simple and efficient method for functional RNA extraction from tropical medicinal plants rich in secondary metabolites
Akshara George*
*Department of Plant Biotechnology, College of Agriculture, Vellayani, Thiruvananthapuram-695522, Kerala, India
Abstract:
Isolation of high-quality functional RNA is prerequisite to facilitate any study related to gene expression and also for downstream applications such as reverse transcription-polymerase chain reaction (RT-PCR), suppression subtractive hybridization (SSH) library construction, differential display (DD), real-time PCR and northern hybridization of medicinal plants. Tropical medicinal plants are rich in polysaccharides, polyphenolics and secondary metabolites that are reported to interfere with the successful RNA isolation. Conventional approaches for the extraction of functional RNA are often time-consuming and need expensive reagents. Moreover, these methods can yield only poor quality and quantity of functional RNA. In our laboratory, we have aimed at establishing a simple and efficient functional RNA extraction procedure. For this, a sodium dodecyl sulfate (SDS) based protocol free of guanidine isothiocyanate was adopted. The total extracted RNA remains in the upper aqueous phase, at acidic conditions leaving DNA and proteins in the lower organic phase. This principle is used in this protocol and the modifications made in the SDS- acid phenol RNA extraction protocol were found to be helpful for extracting RNA from tissues containing large quantities of secondary metabolites. This method can be completed within 3 hours with purity ranges from 1.8–2.0 as confirmed by A260/280 spectrophotometric readings. The above-described RNA extraction protocol works well with all the tissues examined so far, where standard RNA isolation methods failed.
Isolation of high-quality functional RNA is prerequisite to facilitate any study related to gene expression and also for downstream applications such as reverse transcription-polymerase chain reaction (RT-PCR), suppression subtractive hybridization (SSH) library construction, differential display (DD), real-time PCR and northern hybridization of medicinal plants. Tropical medicinal plants are rich in polysaccharides, polyphenolics and secondary metabolites that are reported to interfere with the successful RNA isolation. Conventional approaches for the extraction of functional RNA are often time-consuming and need expensive reagents. Moreover, these methods can yield only poor quality and quantity of functional RNA. In our laboratory, we have aimed at establishing a simple and efficient functional RNA extraction procedure. For this, a sodium dodecyl sulfate (SDS) based protocol free of guanidine isothiocyanate was adopted. The total extracted RNA remains in the upper aqueous phase, at acidic conditions leaving DNA and proteins in the lower organic phase. This principle is used in this protocol and the modifications made in the SDS- acid phenol RNA extraction protocol were found to be helpful for extracting RNA from tissues containing large quantities of secondary metabolites. This method can be completed within 3 hours with purity ranges from 1.8–2.0 as confirmed by A260/280 spectrophotometric readings. The above-described RNA extraction protocol works well with all the tissues examined so far, where standard RNA isolation methods failed.
Fig.: cDNA synthesized from the extracted RNA samples using Revert Aid FIRST strand cDNA synthesis kit (Thermoscientific, USA), Lane M: 1kb plus DNA ladder.
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