A Simple and Rapid System for DNA and RNA Isolation from Diverse Plants Using Handmade Kit

doi:10.1038/protex.2016.015

Method Article

A Simple and Rapid System for DNA and RNA Isolation from Diverse Plants Using Handmade Kit

https://doi.org/10.1038/protex.2016.015

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This protocol describes a rapid DNA and RNA extraction from plant tissues as a handmade kit. Hexadecyltrimethylammonium bromide (CTAB), Sodium chloride (NaCl), Tris base, and Ethylenediaminetetraacetic acid (EDTA) are the main components of this extraction buffer. In contrast to previous works and all reported protocols, the required components of this protocol have established without providing any stock solutions and it will be prepared just by adding them directly. This isolation buffer can extract both DNA and RNA simultaneously. Depending on the purpose of our experiment, RNA and DNA can be selected. Forever, extracting DNA and RNA from plant tissues in a short time by using general laboratory equipment and reach to the high quality of nucleic acid are noticeable. This method probably is usable for all plant tissues and takes only one hour.

Biotechnology

Plant sciences

DNA extraction

Extraction buffer

RNA extraction

Nucleic acid extraction is the purification of DNA and RNA from biological samples which is an important step for the next studies. Studying on the nucleic acid started by Johannes Friedrich Miescher in 1869 who known for discovery of nucleic acid1. He tested a variety of experiments, and finally detected a substance with unexpected properties that called nuclein (today is called DNA). Until now, many procedures were devised for extracting nucleic acid from diverse samples. Chomczynski and Sacchi developed an extraction method based on acid guanidinium thiocyanate phenol chloroform which is universally used2. Allen et al. introduced a modified cetyltrimethylammonium bromide (CTAB) protocol for DNA isolation3. Ghawana et al described an RNA isolation system free of guanidinium salt for plant tissues that have high secondary metabolites4 and other protocols which are usable for specific samples sources. Each of them has its own advantages and disadvantages and is used for a specific range of tissues and organs. Among this several protocols, the CTAB protocol, which was developed by Murray and Thompson has wide applications5. This method is appropriate for the isolation of nucleic acid from plants and is particularly suitable for the elimination of polysaccharide and polyphenolic compounds which reduce the quality of nucleic acid6. Now, CTAB as an important plant DNA and RNA isolation system is routinely used for many purposes.

In this experiment, which lasted few years, after many nucleic acid isolation via several known protocols and using the numerous famous extraction kits with their own unique properties, the modern system was programmed someway that only a strategic reagents be used and prohibited the use of unnecessary materials for preparing the extraction solution. Thus, it has developed a high throughput protocol which can be employed for a widespread range of plant tissues that can compete with commercial kits. This protocol was tested with different samples like apple, pear, potato, thyme, wheat, rice, and many other samples. This method is easy and cost-efficient than all DNA and RNA extraction protocols and commercial kits that have been developed until now. This kit also prepares at minimum period of time without any stock preparation. The purpose of introducing this procedure was accelerating nucleic acid extraction from diverse plants which takes within one hour.

• Hexadecyltrimethylammonium bromide (CTAB; Sigma-Aldrich, cat. no. H5882)

• Tris (hydroxymethyl) aminomethane (Tris base; Sigma-Aldrich, cat. no. 77-86-1)

• Ethylenediaminetetraacetic acid disodium dehydrate (EDTA; Duchefa Biochemie, cat. no. 6381-92-6)

• Sodium chloride (NaCl; Duchefa Biochemie, cat. no. 7647-14-5)

• 2-mercaptoethanol (βME; Sigma-Aldrich, cat. no. 60-24-2) ! CAUTION the βME is caustic, avoid contact by using it under a fume hood and wear protective gloves, lab coat and eye protection.

• Polyvinylpyrrolidone (PVPP; Sigma-Aldrich, cat. no. 9003-39-8)

• Isopropanol (ROMIL Pure Chemistry; cat. no. 67-63-0)

• Chloroform (ROMIL Pure Chemistry, cat. no. 67-66-3) ! CAUTION the chloroform is toxic and produces fumes. Use it under a fume hood and wear protective gloves, lab coat and eye protection.

• Isoamyl alchohol (Acros Organics, cat. no. 30899-19-5) ! CAUTION The isoamylalchohol is caustic. Use it under a fume hood and wear protective gloves, lab coat and eye protection.

• Ribonuclease A (RNase A; Thermo Fisher Scientific, cat. no. 12091-039)

• Ethanol absolute (EtOH; Bidestan Co, cat. no.64-17-5)

• NanoDrop ND-2000 spectrophotometer (Thermo Scientific, cat. no. ND-2000)

• Mortar and pestle

• Water bath

• Fume hood

• PH meter (Brinkmann Metrohm 744 pH Meter 1.744.0010)

• Refrigerating benchtop centrifuge (Heraeus Biofuge Fresco, # 1502)

• Micropipetts (Eppendorf Research 2100, 0.5-10 µl, 10-100 µl, 100-1000 µl)

• RNase-free tubes, 1.5 and 2 ml (FarayandDanesh Arian Co)

Extraction buffer preparation

Add CTAB (0.5 g), EDTA (1 g), Tris base (2.5 g), and NaCl (5 g) as tetrad components of the extraction buffer to 100 ml autoclaved water and then gradually dissolve it by shaking at room temperature.

CRITICAL Add 10 µl βME to 1 ml of the extraction buffer before use to decrease the probably oxidation only for tissues with high polysaccharides and secondary metabolites. CRITICAL add 15 mg PVPP per 1 ml of extraction buffer only for tissues with high polyphenolic pollution.

Homogenization of Tissues TIMING 10 min for 5 samples

1 Ground samples (leaf, shoot, root, and recalcitrant samples, approximately 0.5-1 g) with 1 ml extraction buffer without liquid nitrogen in mortar and pestle that was sterilized.

CRITICAL STEP The procedure are carried out at room temperature except centrifugation steps (at 4℃) and after precipitation of nucleic acid using the isopropanol (on ice).

CRITICAL STEP severely disrupt the tissues to create the glaze mode of samples.

2 Transfer the resulting solution to a sterile centrifuge tube (size=2 ml), and then mix sample by briefly vortexing until the sample is thoroughly resuspended.

Triple Phase Separation TIMING 25 min

3 In this step, incubate samples at 65℃ for 10 min for lysing cells completely.

4 Add 600 µl of chloroform: isoamyl alcohols (24:1) to each tube, homogenize them by vortexing.

5 Centrifuge at 13700 g at 4℃ for 10 min.

CRITICAL STEP Upper phase with extraction buffer at PH=8.5-9 has both DNA and RNA together, while we reduce the PH to 6-7, DNA precipitates and only RNA remains at upper phase.

Precipitation of Nucleic Acid TIMING 15 min

6 Transfer the upper aqueous phase to a new tube (size= 1.5 ml).

7 Add 700 µl of cold isopropanol to precipitate RNA or DNA and then invert tubes 3-4 times to mix the solution.

8 Centrifuge tubes at 13700 g at 4℃ for 10 min. The white pellet will be visible on the bottom of the tubes.

CRITICAL STEP Do not disturb down phases of solution when you pipet the upper phase.

Purification of Nucleic Acid TIMING 5 min

9 Discard the supernatant and wash the precipitate nucleic acid gently with 70% EtOH.

10 After centrifuging at 5400 g at 4˚C for 5 min, remove the supernatant and then air-dry the resultant pellet.

Dissolving and Storage Condition TIMING 5 min

11 Dissolve the pellet in 20-30 µl of RNase free water (commercial) or autoclaved water.

12 After incubate at room temperature for a few minutes, put solubilized nucleic acid in −20˚C for the short time storage or in −80˚C for longtime storage.

Figure 1 shows a schematic model of all the DNA and RNA isolation steps of this procedure.

TIMING

Tissue grinding, Steps 1-2: 10 min

Isolation of nucleic acid, Steps 3-5: 25 min

Nucleic acid precipitation, Steps 6-8: 15 min

Preparation and dissolving the nucleic acid, Steps 9-12: 10 min

?TROUBLESHOOTING

Troubleshooting advice can be found in Table 1.

The main purpose of this research was access to easiest protocol which is prepared as a complete reagents required for nucleic acid isolation. Dual applications of the extraction buffer which result both DNA and RNA that is shown in Figure 2, can be used for DNA purpose by adding only RNase enzyme and for RNA purpose just by adding DNase enzyme to extracted nucleic acid. It is considerable that all of the buffer components consist of weighty scale that is one the big advantages of this protocol. PCR, RT-PCR, real time-PCR and southern blot analysis were implemented for approving the high quality and quantity of isolated DNA and RNA using this protocol which is illustrated in Figure 3.

1 Dahm, R. Discovering DNA: Friedrich Miescher and the early years of nucleic acid research. Human genetics 122, 565-581 (2008).

2 Chomczynski, P. & Sacchi, N. The single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on. Nature protocols 1, 581-585 (2006).

3 Allen, G., Flores-Vergara, M., Krasynanski, S., Kumar, S. & Thompson, W. A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nature protocols 1, 2320-2325 (2006).

4 Ghawana, S. et al. An RNA isolation system for plant tissues rich in secondary metabolites. BMC research notes 4, 85 (2011).

5 Murray, M. & Thompson, W. F. Rapid isolation of high molecular weight plant DNA. Nucleic acids research 8, 4321-4326 (1980).

6 Querci, M., Jermini, M. & Eede, G. The analysis of food samples for the presence of genetically modified organisms. World Health Organization-Joint Research Centre, European Commission Directorate General, Luxembourg (2006).

7 Perry, R., La Torre, J., Kelley, D. & Greenberg, J. On the lability of poly (A) sequences during extraction of messenger RNA from polyribosomes. Biochimica et Biophysica Acta (BBA)-Nucleic Acids and Protein Synthesis 262, 220-226 (1972).

The authors wish to thanks Dr Ali Akbar Habashi and Dr Maryam Jafarkhani Kermani for their critically comments to prepare this protocol.

The authors declare that they have no conflict of interests

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Journal Publication

published 14 Mar, 2016

Read the published version in Protocol Exchange →

Version 1

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A Simple and Rapid System for DNA and RNA Isolation from Diverse Plants Using Handmade Kit

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Journal Publication

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Procedure

Timing

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References

Acknowledgements

Additional Declarations

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A Simple and Rapid System for DNA and RNA Isolation from Diverse Plants Using Handmade Kit

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Reagents

Equipment

Procedure

Timing

Troubleshooting

Anticipated Results

References

Acknowledgements

Additional Declarations

Status:

Journal Publication

Version 1

Privacy Policy

Terms of Service

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