Dataset Information

High-quality RNA isolation from pigment-rich Dendrobium flowers.

ABSTRACT: Isolation of high-quality RNA from Dendrobium flowers is challenging because of the high levels of pigment, polysaccharides, and polyphenols. In the present study, an efficient CTAB method for RNA extraction from the pigment-rich flowers of Dendrobium was optimised. The optimised method yielded high quantities of RNA (10.1-12.9 µg/g). Spectrophotometric values of A260/280 in the range of 2.2 to 2.4 and A260/230 values of 2.0 suggested that the isolated RNA was free of polyphenols, polysaccharides, and protein contaminants. RNA integrity numbers determined by microfluidics were in the range of 7.9-8.9 indicative of intact RNA. In the improved method, the addition of 3 M NaCl and 3% PVP-10 in the extraction buffer, followed by an incubation period of 45 min at 65 °C, eliminated most of the polysaccharides, polyphenolic compounds, and denatured protein. Extraction with phenol:chloroform:isoamyl alcohol (125:24:1) effectively removed pigments from the aqueous phase, while the precipitation of RNA with lithium chloride minimised the co-precipitation of protein, DNA, and polysaccharide and resulted in the extraction of high quality of RNA. The suitability of the RNA for downstream processing was confirmed via RT-PCR amplification of Chalcone synthase gene from cDNA prepared from RNA isolated from different developmental stages of the flower of a Dendrobium hybrid. The present method will be highly useful for the isolation of RNA from pigment, polyphenol, and polysaccharide-rich plant tissues.

SUBMITTER: Khairul-Anuar MA

PROVIDER: S-EPMC6761217 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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High-quality RNA isolation from pigment-rich Dendrobium flowers.

Khairul-Anuar Muhammad-Asyraf MA Mazumdar Purabi P Lau Su-Ee SE Tan Tian Tian TT Harikrishna Jennifer Ann JA

3 Biotech 20190925 10

Isolation of high-quality RNA from Dendrobium flowers is challenging because of the high levels of pigment, polysaccharides, and polyphenols. In the present study, an efficient CTAB method for RNA extraction from the pigment-rich flowers of Dendrobium was optimised. The optimised method yielded high quantities of RNA (10.1-12.9 µg/g). Spectrophotometric values of A260/280 in the range of 2.2 to 2.4 and A260/230 values of 2.0 suggested that the isolated RNA was ...[more]

PMID: 31588395

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Project description:Laser microdissection microscopy (LMM) has potential as a research tool because it allows precise excision of target tissues or cells from a complex biological specimen, and facilitates tissue-specific sample preparation. However, this method has not been used in mosquito vectors to date. To this end, we have developed an LMM method to isolate midgut RNA using Aedes aegypti.Total RNA was isolated from Ae. aegypti midguts that were either fresh-frozen or fixed with histological fixatives. Generally, fresh-frozen tissue sections are a common source of quality LMM-derived RNA; however, our aim was to develop an LMM protocol that could inactivate pathogenic viruses by fixation, while simultaneously preserving RNA from arbovirus-infected mosquitoes. Three groups (10 - 15 mosquitoes per group) of female Ae. aegypti at 24 or 48-hours post-blood meal were intrathoracically injected with one of seven common fixatives (Bouin's, Carnoy's, Formoy's, Cal-Rite, 4% formalin, 10% neutral buffered formalin, or zinc formalin) to evaluate their effect on RNA quality. Total RNA was isolated from the fixed abdomens using a Trizol® method. The results indicated that RNA from Carnoy's and Bouin's fixative samples was comparable to that of fresh frozen midguts (control) in duplicate experiments. When Carnoy's and Bouin's were used to fix the midguts for the LMM procedure, however, Carnoy's-fixed RNA clearly showed much less degradation than Bouin's-fixed RNA. In addition, a sample of 5 randomly chosen transcripts were amplified more efficiently using the Carnoy's treated LMM RNA than Bouin's-fixed RNA in quantitative real-time PCR (qRT-PCR) assays, suggesting there were more intact target mRNAs in the Carnoy's fixed RNA. The yields of total RNA ranged from 0.3 to 19.0 ng per ~3.0 × 10(6) ?m2 in the LMM procedure.Carnoy's fixative was found to be highly compatible with LMM, producing high quality RNA from Ae. aegypti midguts while inactivating viral pathogens. Our findings suggest that LMM in conjunction with Carnoy's fixation can be applied to studies in Ae. aegypti infected with arboviruses without compromising biosafety and RNA quality. This LMM method should be applicable to other mosquito vector studies.

Dataset Information

High-quality RNA isolation from pigment-rich Dendrobium flowers.

Publications

High-quality RNA isolation from pigment-rich <i>Dendrobium</i> flowers.

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