What are the 4 steps of RNA extraction?

Introduction

DNA extraction methods cannot be directly applied to RNA as RNA is structurally very different from DNA. RNA is single-stranded, while DNA is mostly double-stranded. It is often difficult to isolate intact RNA. RNases, a group of enzymes that degrade RNA molecules, are abundant in the environment, including on hands and on surfaces and it is difficult to remove/destroy RNases completely. RNA isolation therefore requires cautious handling of samples and good aseptic techniques. It is important to use only RNase-free solutions during the extraction, as well as RNase-free pipet tips and glassware.

RNA storage and stability

RNAlater solutions from Thermo Fisher and QIAGEN are used by many researchers during RNA isolation, both for stabilizing cellular RNA in tissue samples, and for stabilizing final purified RNA. Ambient storage of tissue samples in RNAlater preserves RNA integrity similarly as storage at low temperature [4, 5]. RNAlater is based on the inhibition of RNAses by sulfate salts such as ammonium sulfate at specific pH [6]. RNA quality can be checked using agarose gel electrophoresis. For long-term storage, RNA should be kept at -80°C in single-use aliquots, while the ethanol precipitate of RNA can be stored at -20°C.

Table 1. RNA extraction methods and typical kits.

Determining RNA quality and quantity

RNA quality can be determined by examining the ratio of absorption at 260 nm and 280 nm with UV spectrophotometry. For high-quality RNA, A260/A280 ratio should be in the range of 1.9–2.1. RNA can be quantified by measuring the absorption at 260 nm, where 1 absorbance unit is equal to 40 μg/ml, at a pH of about 7.5. The validity of this ratio as a measurement of RNA purity was challenged [7]. In addition, the quality of total RNA preparations should be examined through electrophoresis, where both 18S and 28S RNA bands should be very prominent, with the 28S RNA band about twice as intense as the 18S band. Qubit assays, provided by ThermoFisher Scientific, can also quantity RNA samples and assess their quality. Deming et al examined the quality of RNA preparations from parietal lobe tissues of postmortem frozen human brains with RNA 6000 Pico Assay using Bioanalyzer 2100 from Agilent Technologies [8]. Garrett-Bakelman FE et al used RNA Nano kit from Agilent on a Bioanalyzer 2100 from Agilent to examine the quality of RNA preparations from NASA twin astronaut blood samples [9].

Table 2. Basic steps involved in the RNA extraction using organic solvents/chaotropic agents.

RNA Extraction Methods

The major categories of RNA extraction methods are listed in Table 1. Table 2 lists the basic steps involved in the RNA extration using organic solvents/chaotropic agents. Other methods utilize some of the steps or similar steps.

Figure 1. Comparing 14 commercial kits for DNA and RNA extractions from FFPE tissues [1].

RNA extraction from plants

Methods for RNA extraction need to be tailored to the organism from which the RNA is being extracted. Plants pose additional challenges due to the presence of secondary metabolites, polyphenols and polysaccharides. The addition of polyvinylpyrrolidone (PVP) to the extraction buffer was shown by Chan et al to aid the removal of phenolic compounds and polysaccharides from mangosteen leaves and flowers [11]. Birtic et al used an optimized concentration of PVP, along with DNase and ethanol precipitation, to isolate high-quality RNA from seeds of five plant species [12], while Xie et al developed modified RNA extraction methods to obtain high-quality RNA from cotton roots [13]. Tattersall et al evaluated 15 methods/kits, and found that tris-lithium chloride and RNeasy Midi+polyethylene glycol gave the best quality RNA from Grapevine leaves — they published their results in American Journal of Enology and Viticulture [14]. Commercially available RNA isolation kits tailored for plants are available, such as Spectrum plant total RNA kit from MilliporeSigma [15-17] and RNeasy plant kits from QIAGEN [15, 18-25] and Macherey-Nagel NucleoSpin RNA Plant Kit [26].

Figure 2. Blood collection tubes and extraction kits were evaluated for mRNA and MiRNA profiling [2].

Comparison Studies

In order to extract small RNAs from blood and cerebrospinal fluid (CSF), Burgos et al compared ten commercially available RNA-isolation kits: MaxRecovery BiooPure RNA Isolation Reagent (BiooPure; BiooScientific), mirVana miRNA Isolation Kit (mirVana; Thermo Fisher), mirVana PARIS (PARIS; Thermo Fisher), TRI Reagent RT (MRC RT; Molecular Research Center), TRI Reagent RT-Blood (MRC RT-B; Molecular Research Center), TRI Reagent RT-Liquid Samples (MRC RT-LS; Molecular Research Center), RNAzol (Molecular Research Center), miRNeasy (QIAGEN), PureLink microRNA Isolation Kit (PureLink; Thermo Fisher), and mirPremier (MilliporeSigma), and found that mirVana PARIS (Thermo Fisher), mirVana (Thermo Fisher), miRNeasy (QIAGEN), and BiooPure were the best, based on small RNA yield [27].

Glycogen can be added during Trizol extraction to facilitate precipitation of small RNAs and to increase RNA recovery [28, 29].

For gene expression studies in small biopsy tissue samples from the equine gastric epithelium, KAPA SYBR qPCR Master Mix (2x) Universal was found to be superior over SYBR® green mixes from qPCRBIO, QuantiNova, or PerfeCTa and the RNA extraction kit GENEzol is better than TRIzol™ or MiniPrep [30].

Patel et al compared the yield and quality of RNA and DNA from 0.6-mm FFPE prostate tissue cores with 14 commercially available kits, and concluded that QIAGEN AllPrep kit is most suitable for FFPE tissue (see figure 1) [1].

A modified RNAzol protocol yielded more abundant amplifiable microRNA as compared to the standard RNAzol protocol [31].

For the extraction of hepatitis C virus RNA in sera from individuals with hyperlipidemia, hyperbilirubinemia and hyperglobulinemia for RT-PCR analysis, silica nanoparticles were found to be superior over both Trizol and guanidine isothiocyanate methods [32].

Figure 3. The result of an evaluation of six RNA extraction kits [3].

Magnetic bead-based RNA extraction modalities were compared [33]. Silica-coated beads for total RNA extraction, oligo (dT) beads for mRNA extraction, and beads functionalized with oligonucleotides complementary to respiratory syncytial virus (RSV) nucleocapsid gene, respectively recovered ∼75, ∼71, and ∼7% target RSV mRNA after a 1 min of incubation time [33]. RSV-specific beads required much longer incubation times to recover amounts of the target comparable to the other beads (∼77% at 180 min) [33]. The silica-coated beads are suitable for applications that require nucleic acids other than mRNA, especially with detection strategies that are tolerant of a high concentration of nontarget background nucleic acids, such as RT-PCR [33].

RNA quantity and quality were evaluated for mRNA and miRNA profiling with PAXgene and Tempus blood collection tubes and six kits: QIAGEN PAXgene Blood miRNA Kit, Thermo Fisher MagMAX for Stabilized Blood Tubes RNA Isolation Kit, and Norgen Biotek Preserved Blood RNA Purification Kit I and Kit II, and QIAGEN QIAsymphony and Thermo Fisher MagMAX Express-96 Magnetic Particle Processor (see figure 2) [2].

RNAqueous kit (Thermo Fisher), Micro-to-midi total RNA purification system (Thermo Fisher), NucleoSpin RNA II (BD), GenElute mammalian total RNA kit (MilliporeSigma), RNeasy mini kit (QIAGEN), and TRIzol LS reagent (Thermo Fisher) were evaluated for RNA extraction on blood, tonsil, liver, kidney, spleen, bladder, heart, submandibular lymph node from pig infected with classical swine fever virus (CSFV). All extraction kits yielded RNA for positive results in RT-PCR for CSFV, and conventional RT-PCR detected both CSFV and porcine beta-actin in all RNA preparations (RNA extracted from blood by TRIzol LS reagent needed to be 1:2, 1:4, or up to 1:64 in nuclease-free water). TRIzol LS reagent constantly had the highest yield and RNAqueous kit had the highest A260/A280 ratio. NucleoSpin RNA II and GenElute mammalian total RNA kit removed DNA contaminants better [34].

When compared to guanidine isothiocyanate-based techniques, the silica-membrane-based method yielded similar quality and quantity from both fresh and frozen mouse lung tissues and conserved better the intactness of ribosome RNA and short-, medium-, and longer-sized RNA fragments [35].

Figure 3 shows the result of another evaluation of six RNA extraction kits, indicating that all non-phenol-based kits yielded high-quality RNA and one of them AxyPrep Multisource Total RNA Miniprep Kit eliminated genomic DNA contamination completely and in general was much superior over other five kits [3].

Table 3. Commonly used reagents, methods or kits for RNA extraction among formal articles surveyed by Labome. Major suppliers are listed. Num: the number of articles.

RNA Isolation Reagents and Kits in the Literature

Labome survey results

Labome conducts surveys of randomly selected formal articles citing RNA extraction kits. Table 3 lists reagents, methods or kits that are cited most frequently. The sample types that the kits can process (animal cells, plants, etc.) are shown in Table 4. An overview of the kits including their applications, sample sizes, and particular advantages is presented in Table 5.

Two most commonly used kits are RNeasy kits from QIAGEN and TRIzol from Thermo Fisher. RNase-free DNase is used specifically for RNA purification by removing all contaminating genomic DNA. Although commercial kits are most often used, a few studies still use basic organic solvent extraction methods. For example, Meertens L et al purified viral RNA produced through the mMESSAGE mMACHINE SP6 or T7 Transcription Kit from Thermo Fischer Scientific with phenol:chloroform extraction and isopropanol precipitation [63].

Table 4. Sample types that different kits can process.

Major suppliers for RNA extraction reagents and kits

Thermo Fisher

Over the years, Thermo Fisher obtained many brands providing RNA extraction kits, including Ambion, Applied Biosystems, Arcturus, Invitrogen, and Life Technologies. TRIzol (discussed below in more detail) is the most commonly used Thermo Fisher reagent for RNA extraction. Other Thermo Fisher kits that have been used for RNA isolation include PureLink Plant [59], MagMax-96 Total RNA Isolation Kit (good for samples with low cell numbers) [64], LeukoLOCK total RNA 448 isolation system with MagMax technology [52], Purelink RNA Mini kit [65], Dynabeads mRNA Purification Kit [61] and Arcturus PicoPure RNA isolation kit [66, 67]. For example, Nagamatsu G et al isolated mRNA from mouse oocytes with the Dynabeads mRNA DIRECT Micro Kit from Invitrogen for RNA-seq analysis to investigate the dormancy of immature oocytes in primordial follicles [61]. Wang Y et al obtained RNA from microchip-captured circulating tumor cells for RT-PCR with Arcturus PicoPure RNA Extraction buffer [67].

QIAGEN

RNeasy kits (discussed below in more detail) is the most commonly cited RNA extraction method. Besides RNeasy kits, QIAGEN RNA extraction products cited in the survey include QIAzol (optimized for fatty tissues) [68], RNase-free DNase [69] and AllPrep DNA/RNA Micro Kit [70, 71], miRNeasy kit [10, 37, 72], Oligotex kit [73, 74], mRNA isolation/purification kit [75], RNA protect Bacteria Reagent [76], and QIAamp kits [77, 78]. For example, Abbink P et al extracted RNA from rhesus macaque plasma and viral stock using QIAamp Viral RNA mini kit for viral sequencing [77]. Chopra S et al extracted total RNA for RT-PCR from mouse bone marrow–derived dendritic cells, human monocyte–derived dendritic cells, and leukocytes recovered from mouse peritoneal lavage samples with RNeasy Mini kit or QIAzol lysis reagent [68].

MilliporeSigma

Kits supplied by MilliporeSigma that have been used for RNA isolation include GenElute Mammalian Total RNA Miniprep kit [79], Plant Total RNA Kit [15], Spectrum Plant Total RNA kit [16, 17], and TRI reagents [80, 81]. MilliporeSigma also distributes kits and reagents provided by Roche, such as RNase free DNase and High Pure RNA Isolation kit.

Stratagene/Agilent technologies

RNA isolation kits provided by Stratagene include Absolutely kits [82, 83] and RNA 6000 kits [84, 85].

Promega

RNA isolation kits available from Promega include SV total RNA isolation kit [86-88] and RNase-free DNase [89, 90], and the PolyAtract mRNA isolation system [49].

Macherey-Nagel

NucleoSpin kits [47, 83], nucleobond RNA/DNA 400 column [91] are from Macherey-Nagel. Hyun Y et al purified total RNA, containing small RNA (< 200 nt), from plant tissues by using the NucleoSpin RNA set for NucleoZOL [46].

Other kits available for RNA isolation

D Cervettini et al extracted tRNAs from bacteria with phenol and centrifugation [92]. Stadtmauer EA et al extracted total RNA from human whole marrow using Ribopure blood kits from Ambion [93] Corman VM et al extracted RNA with Roche MagNA Pure 96 system (based on magnetic glass bead technique) from sputum, nose and throat swab clinical samples for real-time RT-PCR to detect 2019-nCoV [94]. Nott A et al collected cells in Trizol and purified RNA with the Zymo kit from Zymo Research [95]. Nam S et al isolated RNA using RNAClean XP from Beckman Coulter (A63987) from fly larval brains and purified polyA mRNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module Kit from NEB (E7490) for RNA-seq to study sex-linked tumor dimorphism [60]. Noda S et al isoloated total RNA from primary dental pulp stem cells with QuickGene-Mini80 from Kurabo for real-time PCR analysis [96]. Litke JL et al purified RNA from in vitro transcription reactions with the RNA Clean & Concentrator kit from Zymo Research (R1015) [97]. Huang H et al employed Direct-zol kit from Zymo and Dynabeads mRNA DIRECT kit from Thermo Fisher Scientific to obtain mRNA from HepG2 cells for m6A-seq [49]. Direct-zol kit is gaining popularity for RNA extraction from bacteria [50] and from cultured cells and tissues [51].

Table 5.Overview of kits including applications and particular advantages.

Detailed Discussion on TRIzol/TRI Reagent and RNeasy Kits

RNeasy

RNeasy combines the selective RNA binding of a silica membrane with a fast microspin to extract RNA from sample tissues. It is simple, safe and fast, but more expensive than guanidine isothiocyanate-based techniques. The silica-membrane-based method seemed to be either comparable to or superior over guanidine isothiocyanate-based techniques in terms of RNA yield, RNA quality, and the preservation of intact short, medium and long RNA molecules [35].

RNeasy kits, including the various version like Lipid Tissue Mini [100], are cited most frequently among the articles surveyed by Labome. Zeng Q et al obtained mRNA from cultured cells for RT-PCR with miRNeasy kit (217004) [38]. Hsueh MF et al used the same kit to extract RNA from frozen cartilage tissues for miRNA quantification by real-time polymerase chain reaction [37]. DiTroia SP et al extracted RNA from mouse primordial germ cells for RT-PCR with the Micro kit [101]. Lardennois A et al purified double-stranded RNAs produced by the Ambion mMessage mMachine kit with the RNeasy MinElute Cleanup kit to investigate the role of mechanical forces in body-axis elongation [36]. Wilson ST et al extracted total RNA from polycarbonate filters collecting seawater microorganisms for metatranscriptome analysis using QIAGEN RNeasy Mini Kit, with the addition of Biospec zirconia/silica beads [69]. Deming et al extracted RNA from parietal lobe tissues of postmortem frozen human brains with TissueLyser LT and RNeasy Mini Kit [8]. Lee J et al isolated total mRNA from iPSC-derived cardiomyocytes for qPCR with QIAGEN miRNeasy Mini kit [72]. Kulkarni S et al extracted RNA from peripheral blood lymphocytes with RNeasy Universal kit for RACE application and qPCR of a lncRNA [39]. Zhao N et al used RNeasy Mini Elute Cleanup Kit from QIAGEN to purify mRNA from in vitro transcribed T7-FB-GFP and T7-N×HA-mCh-H2B using mMESSAGE mMACHINE kit from ThermoFisher Scientific for injection into zebrafish embryos [102]. RNeasy variations include RNeasy Plus Mini Kit [45, 103] and RNeasy Micro kit [104], among others.

TRIzol/TRI Reagent

TRIzol reagent by Thermo Fisher (for example, 15596-018 [105] ) and TRI reagent from MilliporeSigma have been used to extract RNA for a wide range of purposes. Duncan A et al purified RNA for RT-PCR from rat habenula tissues with TRIzol Reagent from Thermo Fisher and chloroform/isopropanol precipitation [44]. Zullo JM et al obtained RNA for qPCR from worms with Trizol and chloroform/ethanol treatment [106]. Liu Y et al extracted total RNA from mouse peritoneal macrophages with TRIzol reagent from Invitrogen for m6A dot blot and qPCR [107]. Rosshart SP et al extracted RNA from mouse red blood cells for RNA-seq with TRIzol from Invitrogen Life Technologies to study microbiota and immune responses in laboratory mice born to wild mice [108].

Thermo Fisher’s TRIzol reagent has been used to extract RNA from a multitude of sources, including frozen splenic tissues [109], transfected Hela and HEK293 cells and mouse brain tissues [110], transfected HEK293 cells [97], and plants [111], among others.

History

Dr. Cox initially introduced guanidinium chloride as a deproteinization agent for RNA isolation in 1968, and over the years it became the favored method for RNA purification. In 1979, Dr. Chirgwin managed to isolate undegraded RNA from pancreas by using guanidinium methods [112]. In 1982, Dr. Feramisco from Cold Spring Harbor Laboratory combined guanidinium with hot phenol for RNA isolation. Dr. Chomczynski in 1986 published a novel approach, which, in a single-step extraction, isolated RNA in 4 hours [113]. The method possessed unique advantages such as short time, high yield, RNA preservation, and large capacity. Dr. Chomczynski and colleagues were granted a patent with the priority date of Jan 28, 1992 (Patent No. US 5346994 A) and the trademark of TRIzol was granted to their organization Molecular Research Center. In the subsequent years, their method and product have been widely used and gained “overwhelming number of citations ... over 20 years” [114]. Molecular Research Center eventually became part of Thermo Fisher, and part of Thermo Fisher in 2013.

Main applications

The TRIzol reagent is mainly used for RNA isolation (including microRNA), DNA isolation and protein isolation from tissues or cells. According to the product manual, isolated RNA can be used in RT-PCR, Northern Blot analysis, Dot Blot hybridization, poly(A)+ selection, in vitro translation, RNase protection assay, and molecular cloning. Isolated DNA can be used in PCR, Restriction Enzyme digestion, and Southern Blots. Isolated protein can be used for Western Blots, recovery of enzymatic activity, and immunoprecipitation.

Important points

The TRIzol method is the most widely used method for RNA isolation or DNA and protein isolation. Both DIY and commercial reagent kits can be effective. For the TRIzol method, variations are provided by Thermo Fisher Corporation (and also other providers). Users should read the product sheets, and make a choice based on the type of samples such as solid or liquid sample, cells or bacteria. TRIzol reagent contains chemicals that are toxic, irritant, and corrosive. Direct contact with TRIzol may cause pain, redness, ulcer and scar on the skin. Thus users should always avoid breathing vapor; avoid skin and clothes contact; always wear disposable gloves, and use eye protection, among other safety precautions. Common problems when using TRIzol reagents include low RNA yield, RNA degradation, inhibition of downstream enzymatic reactions, low A260/A280 ratio, contamination with DNA/RNA/protein. Users should consult the product data-sheet and very often the providers' websites for troubleshooting's. Some of the important issues are 1) storage of reagents and samples; 2) sample lysis and homogenization; 3) sterilization of labware, automatic pipette tips; 4) aspiration in target interface to prevent DNA/RNA/protein contamination; 5) pellet fully redissolving; 6) strict RNase-free environment in downstream sampling; 6) or even clean bench.

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What are the steps for RNA extraction?

How is RNA extracted and purified?

Why do we do RNA extraction?