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Simple and Rapid Isolation of Plant RNA,,, Using the Eppendorf Perfect RNA, Eukaryotic, Maxi Kit

Simple and Rapid Isolation of Plant RNA Using the Eppendorf Perfect RNA, Eukaryotic, Maxi Kit

Sonja Vorwerk and Barbro Patterson
Sonja Vorwerk, Max Planck Institut fr Zchtungsforschung, Carl-von-Linn Weg 10, 50829 Cologne, Germany
Phone: ++49-221-5062-441, Fax. ++49-221-5062-413, e-mail: vorwerk@mpiz-koeln.mpg.de
Barbro Patterson, Eppendorf AG, Germany Abstract

The isolation of plant RNA is a particularly challenging task. There are many protocols for the isolation of total RNA from plants that utilize solvents such as phenol or chloroform; however, their toxicity and the resulting cumbersome disposal may make alternative protocols more desirable. This application protocol demonstrates that the Eppendorf Perfect RNA Maxi Kit for eukaryotic cells is ideally suited for the isolation of total RNA from plant material, shown here with the sources Arabidopsis thaliana (dicotyledon) and Zea mays (monocotyledon).

The technology of the Perfect RNA Kit is based on cell lysis with a chaotropic guanidinium isothiocyanate solution, resulting in the rapid inactivation of cellular RNases. The RNA in the lysate is then selectively bound to a specially designed matrix and purified from DNA and other contaminants by efficient washing steps. Finally, RNA is eluted in molecular biology grade (RNase-free) water.

Materials and Methods

Arabidopsis plants, maize plants, and a maize cell culture were used for the experiments. Various amounts of starting material were used, from 110 mg (see results listed below). First, the plant material was ground very thoroughly to a fine powder in liquid nitrogen for a minimum of 10 min. and then transferred into a fresh mortar (to prevent the buffer from freezing). All further procedural steps were performed according to the protocol of the Perfect RNA Maxi Kit, with a slight modification to Steps 13 (see protocol below). Quantity and quality of the obtained RNA were determined based on their UV absorptions at A260 nm and A280 nm. Two g of each RNA sample eluate were analyzed by agarose gel electrophoresis. RNA samples were concentrated by ethanol precipitation for subsequent RT-PCR amplification of two Arabidopsis-specific gene fragments. Two g of total RNA from an Arabidopsis leaf/flower/pod mixture were used in a 20 l RT reaction. Two l of the RT reactions were used as a template for the 50 l PCR (Two-step RT-PCR). cDNA synthesis was performed with a Thermoscript RT-PCR-System (Invitrogen/Life Technologies) according to the manufacturer's protocol. PCR was performed with Ex Taq (TaKaRa) under the following conditions:

Initial incubation at 96C for 4 min.; 36 cycles:

  1. Denaturation at 94C, 30 sec.
  2. Annealing at 64C, 30 sec.
  3. Elongation at 72C, 2.5 min.
  4. Final incubation at 72C for 5 min.
Protocol
  1. Lyse cells by adding 9 ml lysis solution to the mortar and homogenize for approximately 1 min. using a pestle.
  2. Transfer lysate into a 50 ml centrifuge tube which can withstand 3,000 x g. Add an additional 9 ml of lysis solution and vortex vigorously.
  3. Incubate homogenized cells or tissue for at least 5 min. at room temperature. Centrifuge at 3,000 x g for 20 min. Transfer supernatant pellet to a fresh 50 ml tube without any carryover of the cell debris. If the pellet is loose, the supernatant should be filtered through an RNase-free Miracloth (Callbiochem).
  4. Add 18 ml 70% ethanol to lysate and completely mix via very gentle, repeated inversion.
  5. Add 4 ml well-mixed Perfect RNA Binding Matrix Solution and mix via gentle inversion.
  6. Place two Perfect RNA Maxi Spin Columns into 50 ml centrifuge tubes and transfer lysate/Binding Matrix mixture to two Spin Column assemblies.
    Note: A modified preparation procedure for difficult tissues is described in the detailed protocol Step 6.
  7. Centrifuge for 5 to 10 min. at 2,000 x g. Discard filtrate.
  8. Add 10 ml Wash Solution I to each Spin Column and centrifuge for 5 min. at 2,000 x g. Carefully transfer Spin Columns to fresh centrifuge tubes without splashing Wash Solution I.
  9. Add 10 ml Diluted Wash Solution II (ethanol not provided) to each Spin Column and centrifuge 5 min. at 2,000 x g. Carefully transfer dry Spin Columns to RNase-free 50 ml Collection Tubes (provided).
  10. Add 1 ml Molecular Biology Grade Water to each Spin Column and vortex assemblies briefly.
  11. Incubate at 50C for 5 min.
  12. After incubation, vortex for 5 sec. and immediately centrifuge for 5 min. at 2,000 x g. Store recovered total RNA at 80C.
Results

About 200 g RNA of each were obtained from 1 g source material of Arabidopsis leaves, flowers, or pods, while 5 g yielded about 800 g RNA, and 10 g yielded 850 g RNA. With respect to maize, the yields were somewhat lower: 180 g, 560 g, and 600 g RNA were yielded from 1 g, 5 g, and 10 g leaves, respectively. Two g sprouts yielded 170 g RNA. When 2 g source material from a BMS maize cell line was used (suspension culture), the yield was 600 g RNA. Purity was determined by examining the A260/280 ratio. All samples had ratios between 1.9 and 2.05, demonstrating a high degree of purity. Figure 1 shows that the Perfect RNA isolation procedure produces intact and non-degraded RNA. Figure 2 represents successful RT-PCR amplification of a 2331 bp and an 891 bp-specific gene fragment of Arabidopsis.

Figure 1: Two g total RNA from Arabidopsis leaves (lane 2), flowers (lane 3), and pods (lane 4) were run on a 1% agarose formaldehyde gel and stained with ethidium bromide. Lane 1: 0.24-9.5 kb RNA Ladder from Invitrogen/Life Technologies. RNA had been isolated using the Perfect RNA Maxi Kit. The distinct 18S and 28S rRNA bands demonstrate the excellent total RNA quality. Mitochondrial and chloroplast ribosomal RNA are also visible. Figure 2: Ethidium bromide-stained 1% agarose gel. The gel demonstrates the successful amplification of the 2331 bp Acc. BAB08877 and 891 bp Acc. BAB01090 Arabidopsis gene fragment. RNA was isolated using the Perfect RNA Eukaryotic Maxi Kit. Five l each of the RT reaction had been used for agarose gel electrophoresis. Lane M: 1 kb standard (Invitrogen/LifeTechnologies); Lane 1: amplified 2331 bp fragment; Lane 2: negative control without RNA; Lane 3: amplified 891 bp fragment; Lane 4: negative control without RNA. Discussion

The Eppendorf Perfect RNA Kit for eukaryotic cells is well suited for the isolation of intact total RNA from plants. Within 2.5 hours, highly purified, high molecular weight total RNA can be obtained from various source materials such as leaves, flowers, pods, and plant cell cultures. The method is efficient and eliminates exposure to hazardous organics. Conventional phenol/chloroform extraction with subsequent ethanol precipitation requires almost twice the time. Also, the buffer supplied with the kit significantly reduces the risk of RNase contamination during isolation. 180-800 g RNA can be isolated from 1-5 g plant material. The use of more than 5 g source material does not result in a pronounced increase in yield. When plant cell cultures were used as the starting material, the RNA yield was almost doubled compared to the plant material. The measurement of the A260/280 ratios confirmed the reproducible extraction of highly pure, intact, total RNA. RNA isolated with the Perfect RNA Kit can be directly used in demanding subsequent reactions like RT-PCR.

References

  • Chomczynski P, Sacchi N., Anal Biochem 1987 Apr;162(1):156-9, Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
  • Chirgwin, J.M., et al. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochem. 18(24): 5294-5299.
  • Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. pp.188-190, 196.
  • Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual 2nd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. pp.7.19-7.22.

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