RNASwift: A rapid, versatile RNA extraction method free from phenol and chloroform

Abstract

RNASwift is an inexpensive, versatile method for the rapid extraction of RNA. Existing RNA extraction methods typically use hazardous chemicals including phenol, chloroform and formamide which are often difficult to completely remove from the extracted RNA. RNASwift uses sodium chloride and sodium dodecyl sulphate to lyse the cells and isolate the RNA from the abundant cellular components in conjunction with solid phase extraction or isopropanol precipitation to rapidly purify the RNA. Moreover, the purified RNA is directly compatible with downstream analysis. Using spectrophotometry in conjunction with ion pair reverse phase chromatography to analyse the extracted RNA, we show that RNASwift extracts and purifies RNA of higher quality and purity in comparison to alternative RNA extraction methods. The RNASwift method yields approximately 25 µg of RNA from only 10 8 Escherichia coli cells. Furthermore, RNASwift is versatile; the same simple reagents can be used to rapidly extract RNA from a variety of different cells including bacterial, yeast and mammalian cells. In addition to the extraction of total RNA, the RNASwift method can also be used to extract double stranded RNA from genetically modified E. coli in higher yields compared to alternative methods.

Purification and characterisation of dsRNA using ion pair reverse phase chromatography and mass spectrometry

“…This method termed (RNAsnap™) generated similar quality and yield compared to the commercial guanidium isothiocyanate −phenol/chloroform based methods. In addition phenol/chloroform free methods have also been developed for the efficient extraction of RNA [27] . Therefore, optimising efficient extraction of high quality RNA whilst simultaneously minimising RNA degradation and rapidly assessing RNA quality is important.…”

Analysis of long dsRNA produced in vitro and in vivo using atomic force microscopy in conjunction with ion-pair reverse-phase HPLC

“…22 RNA purification was performed using the RNASwift method as previously described. 23 Full details are provided in ESI. † RNA quantification was performed using a Nanodrop 2000 UV visible spectrophotometer (Thermo Fisher Scientific) using an extinction coefficient of 0.021 (μg mL −1 ) −1 cm −1 which corresponds to 1 A 260 = 46.52 µg ml −1 .…”

“…Following E. coli growth, dsRNA was extracted using conditions to ensure all ssRNA is degraded, prior to purification of the dsRNA using solid phase extraction (SPE). 23 For in vitro transcription of dsRNA the corresponding ssRNAs were in vitro transcribed prior to purification using SPE and formation of dsRNA by incubating equal concentrations of the ssRNA. The analyses of dsRNA using native agarose gel electrophoresis following extraction and purification of dsRNA molecules generated in vivo and in vitro are shown in Fig.…”

Overexpression of microRNA‑506‑3p aggravates the injury of vascular endothelial cells in patients with hypertension by downregulating Beclin1 expression

“…Cells (1×10 5 ) were trypsinized and mixed with 0.5 ml TRIzol (Thermo Fisher Scientific, Inc.) for lysis. Total RNA was then extracted using the phenol chloroform method ( 28 ). The purity of RNA was determined by A260/A280 using ultraviolet spectrophotometry (Nanodrop ND2000, Thermo Fisher Scientific, Inc.).…”