LogSpin: a simple, economical and fast method for RNA isolation from infected or healthy plants and other eukaryotic tissues
© Yaffe et al; licensee BioMed Central Ltd. 2012
Received: 25 October 2011
Accepted: 19 January 2012
Published: 19 January 2012
Rapid RNA extraction is commonly performed with commercial kits, which are very expensive and can involve toxic reagents. Most of these kits can be used with healthy plant tissues, but do not produce consistently high-quality RNA from necrotic fungus-infected tissues or fungal mycelium.
We report on the development of a rapid and relatively inexpensive method for total RNA extraction from plants and fungus-infected tissues, as well as from insects and fungi, based on guanidine hydrochloride buffer and common DNA extraction columns originally used for the extraction and purification of plasmids and cosmids.
The proposed method can be used reproducibly for RNA isolation from a variety of plant species. It can also be used with infected plant tissue and fungal mycelia, which are typically recalcitrant to standard nucleic acid extraction procedures.
KeywordsRNA extraction Infected tissue Plant Fungus Aphids
There are several known methods of RNA extraction. Most require reagents such as phenol-chloroform, which according to the US Environmental Protection Agency, are toxic and have a negative effect on the environment [1–3]. A rapid and less toxic option for RNA extraction is the use of commercial kits, but these are significantly more expensive than traditional phenol-chloroform extraction. The price of the extraction can become a limitation when there is a demand to extract RNA from a large number of samples.
Logemann et al.  described a common RNA extraction protocol which makes use of 8 M guanidine hydrochloride buffer to inhibit ribonucleases (RNases) , supplemented with 20 mM MES hydrate and 20 mM EDTA, and fresh addition of 0.0034% β-mercaptoethanol. In this protocol, proteins are removed by phenol/chloroform/isoamylalcohol (25:24:1). The RNA is precipitated by ethanol or isopropanol with 1 M acetic acid and washed with 3 M sodium acetate (pH 5.2) to dissolve low-molecular-weight RNA and contaminating polysaccharides, leaving the intact RNA as a pellet after centrifugation. The salts are removed by a final wash with 70% ethanol and the RNA pellet is subsequently dissolved in sterile water. This and similar protocols are inexpensive but are time-consuming and RNA yield varies among samples. In addition, the toxicity of the materials to human health and the environment must be considered.
The quality of RNA extracted from plant tissues using the above protocol is generally high. However, this is not the case with plant species that have high phenol or cellulose contents, from which RNA is difficult to extract. RNA is even harder to extract from fungus-infected tissues, showing only variable success, with very low relative yields and quality [6, 7].
In this work, we report on a simple, economical, fast, and relatively non-toxic high-yielding method for RNA extraction, termed LogSpin. This method combines the RNA extraction protocol described by Logemann et al.  with a standard plasmid DNA extraction spin column. We eliminate the need for most toxic reagents and reduce the extraction time, making it possible to handle a large number of samples on the same day. The LogSpin method produces a high yield of clean, high-quality total RNA from tissues of a variety of plant species, including necrotic tissues and fungal mycelia, which are recalcitrant to RNA extraction by common protocols, as well as from insects. We show that the extracted RNA is suitable for cDNA synthesis and reverse transcriptase applications, including semi-quantitative and quantitative real-time RT-PCR.
Results and discussion
RNA extraction using plasmid DNA extraction columns
Spin columns contain a silica resin that selectively binds DNA/RNA, depending on the salt setting and other aspects influenced by the extraction method. Silica-based nucleic acid purification approaches make use of chaotrophic salts that denature proteins (including DNases and RNases) but also denature nucleic acids by disrupting their hydrogen bonding. This leads to selective binding of the nucleic acids to the silica resin in the column, and their effective separation from the rest of the sample. The nucleic acids are then washed with chaotropic salts to remove protein and pigment contaminants, and with ethanol to remove salts. After washing, nucleic acids are eluted from the column with water or low-salt solution, which induce its renaturation and thus eliminates their affinity for the silica resin.
RNA purification from Arabidopsis by plasmid DNA extraction column (pDNA) (QIAprep Spin Miniprep Kit, Qiagen) and RNA collection column (RNA) (RNeasy, Qiagen)
LogSpin extraction demonstrate plant RNA yields similar to those obtained with commercial kits
RNA purification from tomato and Arabidopsis leaves, petunia flowers and Botrytis cinerea mycelium by LogSpin protocol and by commercial kits
31.3 ± 6.2
2.07 ± 0.01
2.27 ± 0.01
26.7 ± 13.3
2.11 ± 0.00
2.33 ± 0.04
25.1 ± 1.8
2.14 ± 0.01
2.30 ± 0.01
21.1 ± 5.9
2.13 ± 0.00
2.05 ± 0.12
16.3 ± 3.8
2.10 ± 0.01
2.28 ± 0.01
15.5 ± 3.6
2.15 ± 0.01
2.29 ± 0.01
37.8 ± 5.5
1.90 ± 0.06
1.30 ± 0.25
14.7 ± 1.0
2.07 ± 0.02
2.02 ± 0.14
13.6 ± 0.5
2.06 ± 0.02
2.21 ± 0.01
31.0 ± 3.4
2.12 ± 0.00
2.14 ± 0.00
83.3 ± 18.9
1.87 ± 0.07
2.05 ± 0.08
LogSpin extraction leads to higher RNA yield from infected plant tissue and fungi than extraction by commercial kits
RNA purification by LogSpin, Tri-reagent (TRI) and Qiagen RNeasy kit (KIT) from uninfected and infected Arabidopsis tissue
RNA yield1 μg/100 mg
Uninfected - LogSpin
15.5 ± 3.6
2.15 ± 0.01
2.29 ± 0.01
Infected - LogSpin
15.8 ± 4.2
2.16 ± 0.01
2.30 ± 0.02
Uninfected - KIT
16.3 ± 3.8*
2.10 ± 0.01
2.28 ± 0.01
Infected - KIT
9.3 ± 1.7*
2.13 ± 0.01
2.22 ± 0.05
Uninfected - TRI
21.1 ± 3.4&
2.13 ± 0.00
2.05 ± 0.07
Infected - TRI
10.3 ± 0.81&
2.12 ± 0.04
2.02 ± 0.05
The LogSpin method can be used for a diverse range of eukaryotic organisms, including fungus-infected plant tissue
RNA purification by LogSpin from a variety of organisms
RNA yield1 μg/100 mg
25.1 ± 1.8
2.14 ± 0.01
2.30 ± 0.01
12.3 ± 1.2
2.06 ± 0.04
2.30 ± 0.04
15.5 ± 3.6
2.13 ± 0.02
2.22 ± 0.05
13.6 ± 0.5
2.06 ± 0.02
2.21 ± 0.01
1.78 ± 0.2
2.46 ± 0.18
2.07 ± 0.03
B. cinerea 4
83.3 ± 19
1.87 ± 0.07
2.05 ± 0.08
64.8 ± 15
2.14 ± 0.06
2.29 ± 0.11
B. tabaci 6
32.5 ± 2.3
1.94 ± 0.06
1.67 ± 0.10
Our protocol can also be used with RNA-extraction protocols that require phenol for membrane lysis, such as RNA extraction from nuclei. The plasmid DNA column can be loaded with the liquid phase obtained from the phenol extraction. In fact, the DNA spin columns can be coupled with any front-end RNA or nucleic acid extraction method that uses ethanol precipitation to purify RNA.
Reverse transcriptase applications
We compared RNA extraction using our protocol on different plasmid mini prep columns (Qiagen QIAprep Spin Miniprep Kit and the HiYield Plasmid Mini Kit from RBC Bioscience, Taipei, Taiwan). From the results, we assume that the columns have the selective ability (low cutoff) to bind RNA nucleic acids preferentially over genomic DNA. Plasmid DNA extraction columns are designed to bind small DNA nucleic acid fragments (plasmids up to 10,000 bp). Finally, real-time PCR on cDNA synthesized from DNase I-treated Arabidopsis and rose RNA extracted by our LogSpin protocol showed consistently high-quality results (Figure 3B, C and Additional File 1). This indicates that the total RNA extracted with the LogSpin protocol is highly pure, remains intact and can be applied for downstream applications.
Benefits of RNA extraction based on plasmid DNA extraction columns
The first advantage of the LogSpin protocol is economical: according to the prices provided by companies that are marketing RNA purification kits in Israel, the range is 3.75 to 12.2 USD per sample. The price of extracting one sample using the LogSpin protocol was 0.98 USD, ca. ~25% of the cost of the least expensive commercial kit. This price includes the solutions, the extraction columns and the eppendorf tubes.
The second advantage is the relative efficiency of this protocol, which makes it suitable for RNA extraction from a large number of samples (12-18) in less than 1 h (after plant tissue grinding). This allows several rounds of RNA production in the same day, providing numerous samples for treatment or use in further applications (eg., DNase I treatment, RT-PCR, for example) at the same time.
The third advantage of this protocol is the absence of phenol/chloroform/isoamylalcohol mixture and β-mercaptoethanol, compounds that are harmful to both humans and the environment (see Additional file 3).
Thus, not only is our RNA extraction method useful for plant tissues, including infected plant tissues, insects and fungal mycelia, which are normally recalcitrant to RNA extraction, it is also economical, simple and quick, and reduces exposure to harmful chemicals in the laboratory, to the benefit of workers' health and the environment.
In this work, we report a simple, economical, fast, and relatively non-toxic high-yielding method for RNA extraction. We demonstrate the use of our method for RNA extraction from a variety of plant, fungal and insect tissues, and its reproducibility for RNA isolation from infected plant tissues, which are difficult to extract by other methods. The LogSpin method produces a high yield of clean, high-quality total RNA that is suitable for cDNA synthesis and reverse transcriptase applications, including semi-quantitative and quantitative real-time RT-PCR.
Plant material and growth conditions
Arabidopsis thaliana ecotype Ws-0 seeds were sown in soil (30% peat, 30% vermiculite, 20% tuff and 20% perlite) and vernalized at 4°C for 2 days before transferring to a controlled environment. Plants were kept in the growth room at 18°C under an 8/16 h light/dark photoperiod using fluorescent lamps (Osram L 36 W/840, Lumilux Cool White, Munich, Germany). Tomato, petunia and rose plants were grown in the greenhouse at 25°C under natural daylight. Barley plants were collected from the field.
Fungal strains, growth and inoculation method
B. cinerea strain B05.10 and A. brassicicola isolated in Israel were grown on potato dextrose agar (PDA, Difco, France) in a controlled-environment chamber at 22°C under illumination with fluorescent and incandescent light, at a photofluency rate of approximately 120 μmol/m2 s and 12 h day length. Conidia were harvested in sterile distilled water and filtered through four layers of sterile gauze to remove hyphae. For inoculation, conidial suspensions were adjusted to 3000 conidia/μl. B. cinerea conidial suspensions were prepared in half-strength filtered (0.45-μm) grape juice. Infected leaves were collected 72 h post-inoculation for RNA extraction.
Chemicals for RNA extraction
Guanidine hydrochloride for molecular biology (≥ 99% pure), MES hydrate, minimum 99.5% titration, and diethyl pyrocarbonate (DEPC) were from Sigma-Aldrich. EDTA was supplied from J.T. Baker (Deventer, The Netherlands), and Na-acetate was purchased from Merck (Serono, Germany). All chemicals were dissolved in DEPC-treated water.
Grinding the plant tissue
Tissue was inserted into a safety-locked eppendorf tube with two glass beads (diameter: 5-10 mm), frozen immediately in liquid nitrogen, ground in Tissue Lyser (Qiagen, CA, USA) to a fine powder and then kept at -80°C for further use. Until addition of the first solution, tissue samples were kept frozen. Afterward, samples were kept on ice.
Common RNA extraction protocol
The extraction protocol was as described by Logemann et al.  with some modifications. Phenol/chloroform/isoamylalcohol (25:24:1) was prepared in a falcon tube 1 day before RNA extraction, sealed, covered with aluminum foil (the solution is sensitive to light) and left overnight in a fume closet. On the day of the experiment, 0.17 μl β-mercaptoethanol was added to 500 μl guanidine hydrochloride buffer (8 M guanidine hydrochloride, 20 mM MES hydrate and 20 mM EDTA made in the fume hood and sterilized), which was then added to 70 to 100 mg ground Arabidopsis leaves in a safety-locked eppendorf tube and mixed well. The supernatant was moved to a new microcentrifuge tube (liquid phase without the glass beads) filled with 400 μl of the lower (liquid) phase only of phenol/chloroform/isoamylalcohol. The suspension was mixed well for ~5 min by vortexing and centrifuged at 4°C, at maximum speed, for 10 min. The upper (aqueous) phase was collected in a new microcentrifuge tube and the same volume of cold isopropanol was added. The suspension was quickly vortexed and centrifuged at 4°C, at maximum speed, for 10 min. The liquid phase was carefully extracted and appropriately discarded, and the pellet was washed with 500 μl 3 M Na-acetate pH 5.2 (a short vortexing helped resuspend the pellet) and centrifuged at 4°C, at maximum speed for 5 min. The pellet was then washed with 70% EtOH (short vortexing to resuspend the pellet) and centrifuged at 4°C, at maximum speed for 5 min. At the end of the washing step, the pellet was dried at room temperature in a laminar flow hood, resuspended in 30 μl DEPC-treated water at 60°C, and left in a 60°C incubator for 10 min. RNA (1000 ng) was treated with DNase I and reverse-transcribed to produce cDNA. RNA yield and purity, before and after DNase I treatment, was measured spectrophotometrically (NanoDrop ND-1000 spectrophotometer, NanoDrop Technologies, Wilmington, DE, USA).
RNA extraction by plasmid DNA extraction column
Guanidine hydrochloride buffer (500 μl; 8 M guanidine hydrochloride, 20 mM MES hydrate and 20 mM EDTA) was added to 70 to 100 mg frozen and ground Arabidopsis leaf tissue in a safety-locked eppendorf tube and vortexed for 5 to 15 s. The liquid phase (without glass beads) was transferred to a new microcentrifuge tube and centrifuged at 4°C, at maximum speed, for 20 min to sediment the ground leaf tissue as described above. The clear supernatant was then transferred to a new microcentrifuge tube containing 1:1 v/v (~250 μl) 96% EtOH, quickly vortexed and then loaded onto the plasmid DNA extraction column (QIAprep Spin Miniprep Kit, Qiagen, Hilden, Germany or HiYield Plasmid Mini Kit, RBC Bioscience). The plasmid DNA extraction column was assembled in a microcentrifuge tube, and centrifuged at 8,000 g for 45 s. The liquid flow-through was appropriately discarded. The column was washed twice: first with 450 μl 3 M Na-acetate, to remove polysaccharides, proteins and pigments, and then with 320 μl 70% EtOH to remove salts. Between and after washes, the column was centrifuged at 8,000 g for 45 s and the liquid flow-through removed. The column was dried by centrifugation at maximum speed for 2 min. For elution of the RNA from the column, 30 to 40 μl of DEPC-treated water at 60°C were added directly to the column membrane, incubated for 2 min at room temperature and centrifuged at 8,000 g for 2 min. To increase RNA yield, this step was repeated with the liquid flow-through. RNA yield and purity, before and after DNase I treatment, were measured spectrophotometrically and by gel electrophoresis.
RNA measurements and quality control
RNA yield and purity were measured by absorbance at OD260, OD260/280 and OD260/230 and analyzed by gel electrophoresis at 100 V in a 1% agarose gel in 1× TAE buffer (40 mM Tris acetate, 1 mM EDTA). Gels were stained by incubation in ethidium bromide for 20 min, followed by washing in TAE buffer for 20 min, and exposure to UV light. RNA integrity was determined with an RNA 6000 nano chip run on an Agilent 2100 Bioanalyzer, with ~250 ng RNA loaded on the chip.
cDNA synthesis and RT-PCR
RNA (1000 ng) was treated with DNase I (New England Biolabs, Ipswich, MA, USA) and 1000 ng of DNA-free RNA was reverse-transcribed to cDNA (EZ-First Strand cDNA Synthesis Kit for RT-PCR, Biological Industries, Beit Haemek, Israel). The products of the reverse transcription (cDNA) were detected by PCR analysis with Act1 and Tub2 primers (Additional file 2: Table S1), which on a cDNA template generate 431-bp and 411-bp products, respectively, while on a DNA template, product fragments are 670 bp and 1000 bp long, respectively. PCR cycles were: denaturation step at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s and elongation at 72°C for 60 s. A final extension step was performed at 72°C for 10 min. cDNA tubes were kept at -20°C and RNA tubes were kept at -80°C.
Quantitative real-time RT-PCR (qRT-PCR) analysis
qRT-PCR was performed with the SYBR master mix and StepOne real-time PCR machine (Applied Biosystems, Foster City, CA). The thermal cycling program was as follows: 95°C for 20 s; 40 cycles of 95°C for 3 s and 60°C for 30 s. Relative fold change of AtPR1 gene normalized to AtPTB1F on samples from infected versus uninfected Arabidopsis leaves was calculated by the 2-ΔΔCt method. The primer sequences are listed in Additional file 4: Table S2.
T-tests were performed only when data was normally distributed and the sample variances were equal. Otherwise Mann-Whitney Rank Sum Test was performed. Significance was accepted at P < 0.05 and is noted in the text or table captions. All experiments shown here are representative of at least three independent experiments with the same pattern of results.
Logemann protocol coupled with spin columns
Reverse transcription polymerase chain reaction
Quantitative real-time RT-PCR
Potato dextrose agar
The study was funded by research grant no. VWZN2556 from the Niedersachsen-Israel Fund to ML and by research grant no. IS-4210-09 to ML from the United States-Israel Binational Agricultural Research and Development Fund.
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