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Extraction of RNA Using the Dellaporta Extraction Method

Extraction of RNA Using the Dellaporta Extraction Method

INTRODUCTION
Objectives
1. To extract RNA from a plant tissue
2. To determine the presence of and analyze RNA by gel electrophoresis

Background

Obtaining a high-quality RNA is the most important aspect of performing a wide range of molecular techniques in the modern studies. For instance, most molecular techniques such as reverse transcription real-time PCR (RT-qPCR), transcriptome analysis using next-generation sequencing, array analysis, digital PCR, northern analysis, and cDNA library construction rely on the purity of the RNA isolates. Most cells contain high levels of proteins, lipids, DNA, polysaccharides, polyphenols, several pigments and other secondary metabolites (Wen & Deng, 2002), which make RNA unusable for downstream work in molecular biology research (Wang, & Vodkin, 2004). Although the new RNA-based methods are highly specific, reproducible and sensitive and characterized by high discriminatory power, rapid processing time and with low costs, they are strongly limited by the presence of inhibitors in plant tissues.
In the natural state, cells contains two types of nucleic acids; Deoxyribonucleic acid (DNA) and the Ribonucleic Acid (RNA). DNA is important as it is the starting point of all proteins within the cell, but it is the RNA that brings about the expression of the proteins coded in the DNA; a large portion coded by DNA is not even transcribed. By studying the RNA, one can determine which proteins are expressed in a cell (Wang, & Vodkin, 2004). This is why Working with RNA has become more important than working with DNA. In order to work with RNA, it has to be isolated from a given cell or tissues and this called RNA extraction. RNA extraction is the purification of RNA from biological samples.
The ubiquitous presence of ribonuclease enzymes in cells and tissues, which can rapidly degrade RNA, complicates the procedures. Several methods are used in molecular biology to isolate RNA from samples, depending on the source of the cells and the reagents available. In this practical, Dellaporta Extraction buffer was used.


MATERIALS AND REAGENTS

1. 1M TRIS
2. 5M Sodium chloride (NaCl)
3. 0.25mM Ethylenediaminetetraacetic acid (EDTA)
4. 14.3M β-Mercaptoethanol (BME)
5. Sterile distilled water (SDW)
6. 10% Sodium Dodecyl Sulphate (SDS)
7. Vortex
8. Incubator
9. Motor and Pestle
10. Liquid nitrogen
11. 70% ethanol
12. 8M Potassium acetate
13. Ice box
14. centrifuge
15. Isopropanol
16. 70% ethanol
17. Paper towels
18. Nuclease free water
19. Eppendorf tubes
20. Hot water bath

PROCEDURE
A. RNA EXTRACTION

i. The Dellaporta Extraction buffer was first prepared by mixing 2.5ml of 1M TRIS, 0.25ml of 5M NaCl, 2.5ml of 0.25 EDTA, 0.245ml of 14.3M β-Mercaptoethanol and topping up with sterile distilled water to a final volume of 25ml.
ii. A plant leaf was plucked from a plant, specifically from the young shoots and placed in the sterile mortar
iii. 0.3g of leaf sample was ground in liquid nitrogen using the pestle and 1ml of Dellaporta Extraction Buffer was added
iv. The homogenate was then transferred to a 1ml Eppendorf tube and vortexed briefly
v. 140μl of 10% SDS was added, vortexed briefly and then incubated at 65˚C for 20 minutes
vi. After incubation, 625μl of 8M Potassium Acetate was added, mixed gently by inverting the tube and left on ice for 10 minutes
vii. The mixture was then centrifuged for 5 minutes at 14000rpm
viii. 1 ml of the resulting supernatant was then collected and transferred to an Eppendorf tube containing 0.6ml of Isopropanol. The tube was inverted gently to mix and left on ice for 10 minutes
ix. The mixture was then spun at 14500rpm for 10 minutes and the Isopropanol poured off carefully to avoid pouring the RNA pellet
x. The pellet was washed with 500μl of 70% ethanol
xi. The pellet was then quick spun for 1 minute at 14000rpm and the supernatant containing ethanol was poured off
xii. The tube was quick spun again and any remaining ethanol was removed using a micropipette
xiii. The pellet was vacuum dried and stored at 4ËšC or -20ËšC for a long time storage
xiv. The next day, the pellet was resuspended in 70μl of sterile distilled water and run on a 1% Agarose gel electrophoresis
xv. The RNA bands were then viewed under the UV Gel Imaging System

B. GEL PREPARATION AND RNA VISUALISATION

1. The gel used for running the electrophoresis was prepared by dissolving 1.00g Agarose in 100ml of 1M TBE in a conical flask.
2. The solution was then sterilized and further dissolved by subjecting it to the microwave for 2 minutes. The solution was left to boil and was covered to avoid spillage.
3. While the gel solution was in the microwave, the mould for the gel was being prepared. This was done by lining the sides of the molding apparatus with the masking tape.
4. The solution was then removed from the microwave, cooled and then 5µl of Ethidium bromide which had been diluted to 0.1M (or 1µl if it is concentrated) was added into the solution.
5. The gel mixture was then poured into the mold and the mold had been inserted into the Electrophoresis tank.
6. The comb was then inserted into the solution so as to create the loading wells before the gel solidified. The comb had to be placed straight and upright.
7. 1M TBE buffer was then added into the electrophoresis tank until it just covered the gel. The tank was then covered.
8. After the gel had solidified, the comb was removed carefully. Then 5µl of the loading ladder was loaded into the wells, at both ends of the gel.
9. Then 10µl of the sample (extracted DNA) was pipette mixed with a small amount of the loading dye and loaded into the respective wells.
10. Some more 1M TBE buffer was then added into the gel tank and covered the gel up to the marked position.
11. The gel electrophoresis was run for 2 hours and at 80Kwh/V
12. After running the electrophoresis, the gel was removed and taken to the Ultra violet reader and imaging system. The gel was photographed for future reference.
13. The size of the DNA (in Kilo base pairs) was then determined using the ladder band pattern.

RESULTS

The bands of the Extracted RNA visualised under UV light after Gel Electrophoresis. Presence of bands indicates the extraction was successful
DISCUSSION

TRIS is, used to maintain a constant pH (8.0) while EDTA protects the RNA from degenerative enzymes called RNase. EDTA works by binding to bivalent cations such as cu++ which are necessary for RNAse activity. 2-Mercaptoethanol is used to eliminate ribonucleases released during cell lysis. Numerous disulfide bonds make ribonucleases very stable enzymes, so 2-mercaptoethanol is used to reduce these disulfide bonds and irreversibly denature the proteins. This prevents them from digesting the RNA during its extraction procedure (Wang, & Vodkin, 2004). Sodium Chloride helps to maintain the pH of the disrupted cell lysate during acid extraction and provides the salt necessary for RNA precipitation.
The detergent Sodium Dodecyl Sulphate (SDS) functions in disrupting the cell wall and internal cellular membranes by disrupting the non-covalent interactions (hydrophobic) in the native cell structure. The function of the Potassium acetate is to neutralize the high pH brought about by the basic Mercaptoethanol-Dellaporta extraction buffer allowing the RNA strands to renature. The potassium acetate also precipitates the SDS from the solution, along with the cellular debris (Wang, & Vodkin, 2004). Isopropanol is used effectively to precipitate nucleic acids, but it is much less effective with proteins. Hence a quick precipitation using Isopropanol can therefore purify RNA from protein contaminants. Ethanol is added so as to help remove the remaining salts and SDS from the preparation.

The velocity of migration of the RNA in an electric field depends on the electric field strength, the net charge on the protein, and the frictional coefficient. The electric force driving the charged molecule toward the oppositely charged electrode is opposed by the viscous drag arising from friction between the moving molecule and the medium. The frictional coefficient depends on both the mass and shape of the migrating molecule and the viscosity of the medium (Wang, & Vodkin, 2004).. The TBE buffer provides optimum pH for the DNA strands and the gel acts as the separating agent, in that, the speed of migration of the DNA strands within the gel is inversely proportional to the size of the strand; hence, the large strands will migrate slowly while the smaller strands will migrate faster.

No9teworthy, it is important to keep the charge constant as this affects the speed of migration of the molecules. The ladder is very important in that it contains DNA of various lengths and sizes, which upon separation; provide a reference in terms of base pairs to the test DNA. The DNA to be identified will be positive in a sample if it coincides with the known base pair band of the ladder.

The function of Ethidium bromide is to enhance the visibility of the DNA strands under Ultra violet light. This is by the fact that it intercalates with the DNA strand hence showing its position under UV light (Wang, & Vodkin, 2004). The voltage applied affects the migration of the stands within the gel in that, the higher the voltage, the shorter the rate of migration and the higher the sensitivity of the separation process.

CONCLUSION

To generate the most sensitive and biologically relevant results, the RNA isolation procedure must include some important steps before, during, and after the actual RNA purification. Isolation of pure, intact and high quality DNA is so crucial for any molecular genetic studies, especially because of high amounts of compounds present in natural tissues that may interfere with subsequent RNA manipulation. The Dellaporta Extraction method is a reliable and reproducible procedure used in the extraction of RNA as it functions in maintaining both the integrity of the RNA and its purity. Gel electrophoresis is a reliable method used in separating the RNA strands extracted and when coupled with the use of Ethidium bromide, it provides a reliable method for confirming the presence of RNA and knowing the size of the bands by referencing from the ladder. Hence, the practical was successful as the objectives were achieved.

REFERENCES

Chang, S., Puryear, J., & Cairney, J. (2003). A simple and efficient method for isolating RNA from pine trees. Plant Molecular Biology Reporter, 11(2), 113-116.
Wang, C. S., & Vodkin, L. O. (2004). Extraction of RNA from tissues containing high levels of procyanidins that bind RNA. Plant Molecular Biology Reporter, 12(2), 132-145.
Wen, X.P., Deng, X. (2002). The extraction of genomic RNA. Biochemistry, 126: 18-21.

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