- Open Access
A simple and efficient method for isolating small RNAs from different plant species
© Rosas-Cárdenas et al; licensee BioMed Central Ltd. 2011
- Received: 28 October 2010
- Accepted: 24 February 2011
- Published: 24 February 2011
Small RNAs emerged over the last decade as key regulators in diverse biological processes in eukaryotic organisms. To identify and study small RNAs, good and efficient protocols are necessary to isolate them, which sometimes may be challenging due to the composition of specific tissues of certain plant species. Here we describe a simple and efficient method to isolate small RNAs from different plant species.
We developed a simple and efficient method to isolate small RNAs from different plant species by first comparing different total RNA extraction protocols, followed by streamlining the best one, finally resulting in a small RNA extraction method that has no need of first total RNA extraction and is not based on the commercially available TRIzol® Reagent or columns. This small RNA extraction method not only works well for plant tissues with high polysaccharide content, like cactus, agave, banana, and tomato, but also for plant species like Arabidopsis or tobacco. Furthermore, the obtained small RNA samples were successfully used in northern blot assays.
Here we provide a simple and efficient method to isolate small RNAs from different plant species, such as cactus, agave, banana, tomato, Arabidopsis, and tobacco, and the small RNAs from this simplified and low cost method is suitable for downstream handling like northern blot assays.
- Prickly Pear
- Prickly Pear Cactus
- Northern Blot Assay
- High Polysaccharide Content
Over the last decade small RNAs (sRNAs) have arisen as key regulators of diverse biological processes in eukaryotic organisms, including for instance development or stress responses, among others (reviewed in: [1–4]). sRNAs are around 20-30 nucleotide (nt) long, and guide regulatory processes at the RNA or DNA level.
The presence of endogenous sRNAs is now reported for many model plants and various non-model species (e.g. [5, 6]), and elucidating the functions for many of these sRNAs will be a challenge in the near future. Two major classes of sRNAs are microRNAs (miRNAs; 21 and 24 nt long) and small-interfering RNAs (siRNAs; 18-24 nt long), with the latter being the most abundant, though, functionally less understood.
Various protocols are available for sRNA isolation from plants (e.g. [6–14]), though most of them are used for model plant species. Normally, these protocols start with total RNA isolation, followed by the isolation or separation of the low molecular weight RNA species (LMW RNA), containing the sRNAs. The most commonly used protocol is based on the extraction of total RNA using TRIzol® Reagent [15, 16] followed by precipitation of LMW RNAs using polyethylene glycol, and finally resulting in RNA species less than 300 nt long. Another protocol for total RNA isolation from tissues with higher contents of polysaccharides is the cetyltrimethylamonium bromide (CTAB) method [7, 8]. These are useful protocols, but sometimes it is possible that these protocols do not work well for other plants species or specific tissues, or become quite labor-intensive due to difficult handling and the need of extra precipitation steps.
This motivated us to investigate whether it would be possible to find a generic protocol to isolate sRNAs, which would also work for plant tissues with a high polysaccharide content. In this report, a sRNA isolation method is presented that works efficiently for different plant species like cactus, banana and tomato fruits, and agave leaves, but also for Arabidopsis and tobacco with less polysaccharide content. The method presented here is not based on the use of the TRIzol® Reagent or commercial columns and omits the total RNA isolation step and, therefore, becomes a simpler and cheaper sRNA isolation method for plants.
Prickly pear (Opuntia robusta) cactus pads and floral buds were collected at INIFAP (Mexican National Institute of Forestry, Agriculture, and Livestock Research) Campo Experimental Norte de Guanajuato, in San Luis de la Paz, Gto., Mexico. Agave leaves were collected at the campus of CINVESTAV, Irapuato, Gto., Mexico. Arabidopsis thaliana (ecotype Ws-3) and Nicotiana tabacum plants were grown under conventional long day growth conditions (22°C, 16 hours of light). Banana and tomato fruits were purchased at the local market. The samples were sliced, ground to a fine powder in a mortar with liquid nitrogen and stored at -80°C until further use.
Buffers and solutions
• LiCl extraction buffer
100 mM Tris-HCl, pH 9.0
100 mM LiCl
10 mM EDTA
• TBE buffer (1x)
0.9 M Tris-HCl, pH 8.0
0.9 M Boric Acid
2 mM EDTA, pH 8.0
• Loading buffer
10 mM EDTA, pH 8.0
1 mg/ml xylene cyanol
1 mg/ml bromophenol blue
• Polyacrylamide stock solution
12.5% polyacrylamide (Acrylamide:bisacrylamide 19:1; Biorad)
0.5× TBE buffer, pH 8.0
7 M Urea
• Denaturing polyacrylamide gel (for one gel)
5 ml of polyacrylamide stock
25 μl of 20% APS (Ammonium persulfate)
5 μl TEMED (N, N, N', N'-Tetramethylethylenediamine)
• Staining solution
0.001% SYBR Gold (Invitrogen)
0.5× TBE buffer, pH 8.0
3 M sodium acetate, pH 5.2
phenol, pH 8.0
chloroform-isoamyl alcohol (24:1; v/v)
phenol-chloroform-isoamyl alcohol (25:24:1; v/v/v)
5 M NaCl solution
40% polyethylene glycol 8000 solution (PEG 8000)
DEPC treated (0.05%) water
For northern blot analysis
• EDC fixation solution (24 ml)
245 μl of 12.5 M methylimidazole, pH 8.0
0.5 g 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)
• Hybridization solution (100 ml)
10 g dextran sulphate
5 ml of 20% SDS
20 ml of 5 M NaCl
5 ml of 1 M Tris-HCl, pH 7.5
• Wash solution
Small RNA extraction
Place 0.1 g of pulverized frozen tissue in a 1.5 ml microcentrifuge tube and add 500 μl of LiCl extraction buffer and 500 μl of phenol pH 8.0.
Shake or mix well using a vortex for 1 min. Place each sample on ice until all samples are ready.
Incubate tubes for 5 min at 60°C.
Centrifuge for 10 min in a microcentrifuge at max speed at 4°C.
Transfer the upper phase to a new microcentrifuge tube and add 600 μl of chloroform-isoamyl alcohol (24:1; v/v).
Centrifuge 10 min at max speed at 4°C.
Transfer the upper phase to a new microcentrifuge tube and incubate for 15 min at 65°C.
Add 50 μl of 5 M NaCl and 63 μl of 40% polyethylene glycol 8000 (w/v) and mix using a vortex for 1 min, followed by incubation on ice for at least 30 min.
Centrifuge for 10 min at max speed at 4°C (Note: the supernatant contains LMW RNA and the pellet consists of HMW RNA and DNA).
Transfer supernatant to a new microcentrifuge tube and add 500 μl of phenol-chloroform-isoamyl alcohol (25:24:1; v/v/v).
Centrifuge for 10 min at max speed at 4°C.
Transfer supernatant to a new microcentrifuge tube and precipitate LMW RNA by adding 50 μl of 3 M sodium acetate pH 5.2 and 1200 μl of absolute ethanol.
Incubate overnight at -20°C.
Centrifuge for 10 min at max speed at 4°C.
Discard supernatant and dry pellet. When dry, resuspend in 20 μl RNAse-free water.
Determine RNA purity and concentration by measuring their absorbance at 230, 260 and 280 nm, and calculate the A260/A280 and A260/A230 ratios.
Small RNA analysis in polyacrylamide gel
Prepare a denaturing 12.5% polyacrylamide gel by mixing all components (see Materials and Methods; vertical electrophoresis gel system; work RNAse-free). Let the polyacrylamide polymerize for at least 30 min and then remove combs, gels may be stored at 4°C. Note: Polymerization time affects the quality of the run, and we have noted that gels prepared two days previous to their use showed improved band definition.
Pre-run the gel(s) in 0.5× TBE buffer (to remove ammonium persulfate residues) for 2 h at 90 V.
Prepare samples. For 2 μg LMW RNA, add 0.3 (v/v) loading buffer (adjust to the same volume in all samples with RNAse-free water). Incubate samples for 5 min at 65°C to denature RNA and immediately place on ice for at least for 1 min.
Before loading each sample in the gel, wash each gel slot with 0.5× TBE using a syringe.
Load the samples in the gel (fill empty slots with loading buffer) and run for around 2 h at 90 V in 0.5× TBE buffer (until bromophenol blue of the loading buffer reaches the end of the gel).
When the electrophoresis run is ready, take the gel out the chamber and stain for 30 min in 15 ml 0.5× TBE buffer with 0.001% SYBR Gold. Afterwards, rinse for 5 min with RNAse-free water.
Visualize the gel under UV light.
Northern blot analysis
The gel may be used for northern blot analysis. In this report, the northern blot analysis was performed following the protocol by Pall and Hamilton (2008)  with modifications. Use a semidry trans-blot system (Biorad) to transfer the gel to a neutral nylon membrane (Hybond-NX, GE Healthcare) in 0.5× TBE buffer for 1 h at 10 V. Air dry the membrane at room temperature, add 12 ml of freshly made EDC fixation solution, incubate the membrane for 30 min at 60°C, and then rinse twice with RNAse-free water. Repeat this fixation step once more. Let the membrane dry and store at -20°C till further use.
Pre-hybridize with 15 ml hybridization solution (containing denatured salm sperm DNA) for 1.5 h at 60°C, followed by replacing the hybridization solution and adding the labelled probe of interest, and incubate for 24 h at 60°C. In this report, two probes were used (5'-AGGGGCCATGCTAATCTTCTC-3' and 5'-AAGAGCTCCCTTCAATCCAAA-3'), each labelled with [γ-32P]ATP to detect the small nucleolar RNA U6 and miRNA159a, respectively.
Wash the membrane twice with wash solution (first for 4 min, and then a second time for 2 min) at room temperature, followed by exposure to a storage phosphor screen for ~48 h at room temperature.
Here we provide a simple and effective method suitable for sRNA extraction from polysaccharide-rich material such as cactus, agave, banana, and tomato tissues, which also works well for less complex plant tissues form e.g., Arabidopsis and tobacco. This modified extraction method gives good yield and quality of LMW RNA species. Moreover, the LMW RNA obtained from the different plant species was successfully used for northern blot assays. Well defined bands were detected when using the miRNA159a and the small nucleolar RNA U6 probes. Therefore, the sRNA molecules that can be obtained with this low-cost short method are suitable for downstream assays like northern blot hybridization, and most likely also for cloning and sequencing of sRNAs.
We thank Dr. Candelario Mondragón-Jacobo at INIFAP Norte de Guanajuato for providing cactus material, Alba Ramos-Olmos for technical advice with northern blot assays, and two anonymous reviewers for their valuable comments. We also thank the Mexican Science Council (CONACyT) for PhD fellowships to FFRC (199450) and to NDF (159754). The work in de Folter laboratory is financed by CONACyT 82826.
- Carthew RW, Sontheimer EJ: Origins and Mechanisms of miRNAs and siRNAs. Cell. 2009, 136: 642-655. 10.1016/j.cell.2009.01.035.PubMed CentralView ArticlePubMedGoogle Scholar
- Chen X: Small RNAs and their roles in plant development. Annu Rev Cell Dev Biol. 2009, 25: 21-44. 10.1146/annurev.cellbio.042308.113417.View ArticlePubMedGoogle Scholar
- Chen X: Small RNAs - secrets and surprises of the genome. Plant J. 2010, 61: 941-958. 10.1111/j.1365-313X.2009.04089.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Voinnet O: Origin, biogenesis, and activity of plant microRNAs. Cell. 2009, 136: 669-687. 10.1016/j.cell.2009.01.046.View ArticlePubMedGoogle Scholar
- Chen D, Meng Y, Ma X, Mao C, Bai Y, Cao J, Gu H, Wu P, Chen M: Small RNAs in angiosperms: sequence characteristics, distribution and generation. Bioinformatics. 2010, 26: 1391-1394. 10.1093/bioinformatics/btq150.View ArticlePubMedGoogle Scholar
- Lu C, Tej SS, Luo S, Haudenschild CD, Meyers BC, Green PJ: Elucidation of the Small RNA Component of the Transcriptome. Science. 2005, 309: 1567-1569. 10.1126/science.1114112.View ArticlePubMedGoogle Scholar
- Carra A, Gambino G, Schubert A: A cetyltrimethylammonium bromide-based method to extract low-molecular-weight RNA from polysaccharide-rich plant tissues. Anal Biochem. 2007, 360: 318-320. 10.1016/j.ab.2006.09.022.View ArticlePubMedGoogle Scholar
- Carra A, Mica E, Gambino G, Pindo M, Moser C, Pe ME, Schubert A: Cloning and characterization of small non-coding RNAs from grape. Plant J. 2009, 59: 750-763. 10.1111/j.1365-313X.2009.03906.x.View ArticlePubMedGoogle Scholar
- Pilcher RL, Moxon S, Pakseresht N, Moulton V, Manning K, Seymour G, Dalmay T: Identification of novel small RNAs in tomato (Solanum lycopersicum). Planta. 2007, 226: 709-717. 10.1007/s00425-007-0518-y.View ArticlePubMedGoogle Scholar
- Chappell L, Baulcombe D, Molnár A: Isolation and Cloning of Small RNAs from Virus-Infected Plants. Current Protocols in Microbiology. 2005, UNIT 16H.2Google Scholar
- Meyers BC, Green PJ: Plant MicroRNAs - Methods and Protocols. 2009, Humana PressGoogle Scholar
- Moxon S, Jing R, Szittya G, Schwach F, Rusholme Pilcher RL, Moulton V, Dalmay T: Deep sequencing of tomato short RNAs identifies microRNAs targeting genes involved in fruit ripening. Genome Res. 2008, 18: 1602-1609. 10.1101/gr.080127.108.PubMed CentralView ArticlePubMedGoogle Scholar
- Itaya A, Bundschuh R, Archual AJ, Joung JG, Fei Z, Dai X, Zhao PX, Tang Y, Nelson RS, Ding B: Small RNAs in tomato fruit and leaf development. Biochim Biophys Acta. 2008, 1779: 99-107.View ArticlePubMedGoogle Scholar
- Hutvagner G, Mlynarova L, Nap JP: Detailed characterization of the posttranscriptional gene-silencing-related small RNA in a GUS gene-silenced tobacco. RNA. 2000, 6: 1445-1454. 10.1017/S1355838200001096.PubMed CentralView ArticlePubMedGoogle Scholar
- Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987, 162: 156-159. 10.1016/0003-2697(87)90021-2.View ArticlePubMedGoogle Scholar
- Chomczynski P, Sacchi N: The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nat Protoc. 2006, 1: 581-585. 10.1038/nprot.2006.83.View ArticlePubMedGoogle Scholar
- Pall GS, Hamilton AJ: Improved northern blot method for enhanced detection of small RNA. Nat Protoc. 2008, 3: 1077-1084. 10.1038/nprot.2008.67.View ArticlePubMedGoogle Scholar
- Verwoerd TC, Dekker BM, Hoekema A: A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Res. 1989, 17: 2362-10.1093/nar/17.6.2362.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.