• Food Science and Preservation
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• v.4 no.1
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• pp.45-51
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• 1997

In our studies on the role of $\beta$-galactosidase in fruit softening, significant difficulty, was encountered in our attempts to extract RNA from persimmon(Diospyros kaki L. cv. Fuyu) fruit due to astringency and tannin content. Initial, unsuccessful RNA extractions involved methods using guanidinium isothiocyanate/CsCl with and without polyvinylpyrrolidone(PVP), phenol/sodium lauryl sulfate(SDS), guanidinium hydrochloride, as well as polysomal RNA purification method that used 0.2 M Tris-HCI (pH 9.0) containing KCI, Mg-acetate, EDTA, $\beta$-mercaptoethanol, and sucrose. A method was devised which employed treatment of fruit with CO2 gas to diminish astringency prior to RNA extraction, followed by extraction of tissue powders with Proteinase K extraction buffer containing PVP and ascorbate at an alkaline pH. This procedure resulted in the removal of tannins and other polyphenolics and extraction of relatively large amount of high-quality RNA suitable for cDNA library construction and polymerase chain reaction(PCR). Futhermore, the procedure does not use the toxic and corrosive chemical guanidinium isothiocyanate or require ultracentrifugation.

• The Plant Pathology Journal
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• v.20 no.2
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• pp.142-146
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• 2004

Woody plant tissues contain great amounts of phenolic compounds and polysaccharides. These substances inhibit the activation of reverse transcriptase and/or Taq polymerase in RT-PCR. The commonly used multiple-step protocols using several additives to diminish polyphenolic compounds during nucleic acid extraction are time consuming and laborious. In this study, sodium sulfite was evaluated as an additive for nucleic acid extraction from woody plants and the efficiency of RT-PCR assay of commercial nucleic acid extraction kits and small-scale dsRNA extraction was compared. Sodium sulfite was used as an inhibitor against polyphenolic oxidases and its effects were compared in RNA extraction by commercial extraction kit and small-scale double-stranded RNA (dsRNA) extraction method for RT-PCR. During nucleic acid extraction, addition of 0.5%-1.5%(w/v) of sodium sulfite to lysis buffer or STE buffer resulted in lighter browning by oxidation than extracts without sodium sulfite and improved the RT-PCR detection. When commercial RNA extraction kit was used, optimal concentrations of sodium sulfite were variable according to the tested plant. However, with dsRNA as RT-PCR template, sodium sulfite 1.5% in STE buffer improved the detection efficiency of Apple chlorotic leaf spot virus (ACLSV) and Apple stem grooving virus (ASGV) in fruit trees, and reduced the unspecific amplifications signi-ficantly. Furthermore, when viruses existed at low titers in host plant, small-scale dsRNA extractions were very reliable.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.139.1-139
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• 2003

Tissues from woody plant contain higher amount of phenolic compounds and polysaccharides, which give inhibitory effects on reverse transcriptase and/or Taq ploymerase. The common multiple-step protocols using several additives to inhibit polyphenoic compounds during nucleic acid extraction are time consuming and laborious. Sodium sulfite (Na$_2$SO$_3$) was used as inhibitor of polyphenolic oxidases in extraction buffer and compare it's effect between commercial RNA extraction kit and small-scale double-stranded RNA (dsRNA) extraction by RT-PCR. During nucleic acid extraction procedure, addition of 0.5％-1.5％ (w/v) sodium sulfite to Iysis buffer or STE buffer resulted in lighter color change than extracts without sodium sulfite and improve the RT-PCR detection. When commercial RNA extraction kit used, optimal concentration of sodium sulfite were variable according to the host plant. However, using dsRNA as RT-PCR template, 1.5％ sodium sulfite in STE buffer improves the detection of both viruses and unspecific amplifications were reduced significantly, Furthermore, when viruses existed at low titers in host plant, small-scale dsRNA extractions were very reliable.

• Journal of Food Hygiene and Safety
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• v.33 no.1
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• pp.71-76
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• 2018

Norovirus is a leading cause of sporadic pathogenic non-bacterial gastroenteritis worldwide. For the detection of norovirus, reverse transcription real-time PCR (RT qPCR) has quickly become a major tool due to its sensitivity and specificity. However, accurate viral RNA extraction methods are essential for RT qPCR analysis. TRIzol reagents are used to extract RNA from biological materials and are therefore widely used for norovirus RNA extraction. In this study, the yield of viral RNA extraction using TRIzol from genogroup II (GII) among the human norovirus genogroup I (GI) and GII, and murine norovirus (GV) depended on the pH of the virus sample solution. The yield of RNA extraction was higher at the alkaline pH than in the acidic region compared with the Ct (threshold cycle) value of the real-time PCR. From the results of this study, it was found that the pH condition is very important for the quantitative analysis of norovirus by extracting GII RNA using TRIzol.

• Journal of Microbiology and Biotechnology
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• v.15 no.3
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• pp.595-602
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• 2005

A one-step real-time quantitative RT-PCR assay in combination with automated RNA extraction was evaluated for routine testing of HCV RNA in the laboratory. Specific primers and probes were developed to detect 302 bp on 5'-UTR of HCV RNA. The assay was able to quantitate a dynamic linear range of $10^7-10^1$ HCV RNA copies/reaction ($R^2=0.997$). The synthetic HCV RNA standard of $1.84{\pm}0.1\;(mean{\pm}SD)$ copies developed in this study corresponded to 1 international unit (IU) of WHO International Standard for HCV RNA (96/790 I). The detection limit of the assay was 3 RNA copies/reaction (81 IU/ml) in plasma samples. The assay was comparable to the Amplicor HCV Monitor (Monitor) assay with correlation coefficient r=0.985, but was more sensitive than the Monitor assay. The assay could be completed within 3 h from RNA extraction to detection and data analysis for up to 32 samples. It allowed rapid RNA extraction, detection, and quantitation of HCV RNA in plasma samples. The method provided sufficient sensitivity and reproducibility and proved to be fast and labor-saving, so that it was suitable for high throughput HCV RNA test.

• Transactions of the Korean Society of Mechanical Engineers B
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• v.35 no.12
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• pp.1309-1316
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• 2011

This paper presents a high-speed RNA microextractor for the direct isolation of RNA from blood lysate using magnetic oligo(dT) beads. The extraction is performed through lateral magnetophoresis, which is induced by a ferromagnetic wire array inlaid. With this RNA microextractor, more than 80% of the magnetic beads could be separated at a flow rate up to 20 ml/h, and the overall extraction procedure was completed within 1 min. The absorbance ratio of RNA to protein(A260/A280) was greater than 1.7, indicating that the extraction technique yields pure RNA. The feasibility of using this technique in reverse transcription polymerase chain reaction procedures was investigated by cDNA synthesis and PCR processes. The results confirmed that the RNA microextractor is a practical device for easy, fast, and high-precision RT-PCR using minimal amounts of reagent.

• Journal of Microbiology and Biotechnology
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• v.24 no.6
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• pp.812-822
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• 2014

Metagenomic analysis of the human intestinal microbiota has extended our understanding of the role of these bacteria in improving human intestinal health; however, a number of reports have shown that current total fecal DNA extraction methods and 16S rRNA universal primer sets could affect the species coverage and resolution of these analyses. Here, we improved the extraction method for total DNA from human fecal samples by optimization of the lysis buffer, boiling time (10 min), and bead-beating time (0 min). In addition, we developed a new long-range 16S rRNA universal PCR primer set targeting the V6 to V9 regions with a 580 bp DNA product length. This new 16S rRNA primer set was evaluated by comparison with two previously developed 16S rRNA universal primer sets and showed high species coverage and resolution. The optimized total fecal DNA extraction method and newly designed long-range 16S rRNA universal primer set will be useful for the highly accurate metagenomic analysis of adult and infant intestinal microbiota with minimization of any bias.

• Annals of Clinical Microbiology
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• v.19 no.1
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• pp.20-23
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• 2016

In the RNA-based study, it is important to extract high-quality RNA. However, RNA extraction from Mycobacterium tuberculosis is problematic due to its thick, waxy cell wall rich in mycolic acid, which renders the cells resistant to lysis. Using TRIzol reagent and several powerful bead-beating steps, a high quantity of RNA was obtained.

• The Plant Pathology Journal
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• v.26 no.3
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• pp.286-288
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• 2010

Plant tissue homogenization using a mortar or mechanical equipment has been the preferred method for obtaining high yields of total RNA; this method, however, is both time-consuming and expensive. Additionally, homogenization may generate excessive endogenous RNases, polyphenolics, and other substances that reduce the quality and quantity of RNA. In this study, we describe the microwave irradiation-assisted RNA extraction (MIRE) technique which, without tissue disruption and homogenization, allows for the cost-effective and rapid generation of intact RNA from apple cane shavings and the reliable detection of apple virus by RT-PCR.

• Mycobiology
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• v.45 no.4
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• pp.434-437
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• 2017

An improved method for extracting high quality and quantity RNA from a jelly mushroom and a dimorphic fungus-Tremella fuciformis which is especially rich in polysaccharides, is described. RNA was extracted from T. fuciformis mycelium M1332 and its parental monokaryotic yeast-like cells Y13 and Y32. The A260/280 and A260/230 ratios were both approximately 2, and the RNA integrity number was larger than 8.9. The yields of RNA were between 108 and $213{\mu}g/g$ fresh wt. Downstream molecular applications including reverse transcriptional PCR and quantitative real-time PCR were also performed. This protocol is reliable and may be widely applicable for total RNA extraction from other jelly mushrooms or filamentous fungi rich in polysaccharides.