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RNA Extraction and Gel

Electrophoresis

Presented by: Muhammad Usman Mughal

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Contents1. Introduction

2. What is RNA?

3. Comparison with DNA

4. Types of RNA

5. Purpose of RNA extraction

6. Methods of RNA extraction

7. Gel electrophoresis

8. Agarose gel electrophoresis

9. Polyacrylamide gel electrophoresis

10.Conclusion

11.References

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What is RNA?

• Ribonucleic Acid (RNA) is a polymeric molecule.

• It is implicated in various biological roles in coding, decoding, regulation, and expression of genes.

• Found in the nucleus and the cytoplasm

• RNA (ribonucleic acid) store and transfer

genetic information in living organisms.

• Three major type of RNA, mRNA (1-5%),

tRNA (10-15%) and rRNA (>80%)

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Comparison with DNARNA DNA

1. 2´ OH group (ribose) 1. 2´ H (deoxyribose)

2. Uracil binds Adenine (A,U,C,G) 2. Thymine binds Adenine (A,T,C,G)

3. Multiple types and roles 3. One biological form

4. Often permanently modified via splicing

4. Permanently modifications are rare (mutation)

5. Usually single-stranded 5. Double stranded

6. Intermolecular binding 6. Double helix structure

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Types of RNA

a) Messenger RNA (mRNA)

b) Transfer RNA (tRNA)

c) Ribosomal RNA (rRNA)

d) Regulatory RNAs

e) In RNA processing

f) RNA genomes

g) In reverse transcription

h) Double-stranded RNA

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Purpose of RNA Extraction

• To measure the size – examine differential splicing

• For sequence – predict protein product

• Abundance of RNA – measure expression levels

• Dynamics of expression – temporal, developmental, tissue specificity

• Real-time quantitative PCR (qPCR)

• RNA Cloning

• Reverse transcription

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Extraction of RNACTAB Method

• Lysis the cell and all the organelles, to free nucleic acid

• Collect the intact mass of nucleic acid by shaking (vortex or centrifuge)

• Dnase used to eliminate DNA and separate RNA

Materials

Before use add 1% beta marcaptoethanol.

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Procedure

• For tissue lysis, plant materials or fungal spores, hyphae are broken down into small parts by grinding in pestle and mortar adding liquid Nitrogen.

• Then 1% CTAB is added to break down tissue. Then it is incubated at 55-65C for 30 minutes to overnight.

• The incubation is given by increasing temperature. The eppendorf is taken and added 1% CTAB buffer half and tissue material half.

• Then it is centrifuged at maximum speed for 10-15 minutes.

• All the cell debris or imparities settled down at the bottom after centrifugation and upper liquid is formed which is called supernatant.

• Supernatant was shifted to another test tube and mixed with equal amount of chloroform : Isoamyl alcohol.

• Then sample was vortex for 3-5minutes then centrifuged at maximum speed for 10 minutes.

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• The pellet settles down at bottom and supernatant is shifted to another Eppendorf.

• Added equal amount of Iso-propanol and incubated at -20oC for 5 minutes.

• Then is again centrifuged at maximum speed Nucleci acid (DNA/RNA) settle down at the bottom.

• Then it is washed with 70% ethanol and centrifuged again pellet down at the bottom.

• Continue with the RNeasy kit from Qiagen dissolve the pellet in water, add RTL and EtOH and then follow the kit instructions.

• Transfer the liquid into a new eppendorf.

• Add LiCl and mix well. This will precipitate the RNA only.

• Precipitate the RNA for at least 20 min at -20 °C.

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• Spin down at max speed for 30 min at 4 °C

• Keep the pellet, discard supernatant. The pure RNA pellet might be transparent and hardly visible.

• Add 70% ethanol and mix well to wash of the salts.

• Spin down at max speed for 2 min

• Keep the pellet, discard supernatant

• Dissolve pellet in water for 15 min at 65 °C.

• Transfer the liquid into a new eppendorf.

• Store RNA at

-04 °C for a week

-20 °C for a month

-80 °C for a year

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TRIzol Method• This is a modification of the procedure originally described by

Chomczynski P. and Sacchi N. 1987.

Material and Reagents

1) TRIzol Reagent (Commercially available from many venders)

Recipe for 1 L 

Reagents 

Final Concentration 

 Phenol in saturated buffer 380 mL

 38%

Guanidine thiocyanate 118.16 g  0.8 M

Ammonium thiocyanate 76.12 g  0.4 M

Sodium acetate pH 5.0 33.4 mL of 3 M stock

 0.1 M

Glycerol 50 mL  5%

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• 2) 0.8 M sodium citrate / 1.2 M NaClO

• 3) Isopropanol (2-Propanol)

• 4) Chloroform

• 5) DEPC-Water

• 6) 75% ethanol prepared with DEPC-Water

• 7) RNase Inhibitor (e.g., aseERASE TM BIO 101 Cat. 2601-104)

• 8) 50 mL sterile plastic screw-cap centrifuge tubes

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Procedure1. Grind 1g tissue in liquid nitrogen in a mortar and pestle.

2. Transfer powdered tissue to a sterile plastic screw-cap centrifuge tube containing TRIzol reagent. Incubate samples at room temperature for 5 min.

3. Homogenize tissue with homogenizer for 15 seconds.

4. Centrifuge samples at 12000 rpm and 4°C for 10 min.

5. Transfer supernatant into new sterile sterile plastic screw-cap centrifuge tube. Discard pellet.

6. Add chloroform tube and shake it vigorously with vortex for 15 sec.

7. Let tube placed at room temp 2-3 min. Centrifuge tube at 10,000 rpm at 4°C for 15 min.

8. Carefully pippet aqueous phase into a clean screw-cap centrifuge tube, discard interphase and lower phase into waste.

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9. Precipitate RNA by adding isopropanol and sodium citrate half volume of the aqueous phase. Cover tube and mix by gentle inversion. Let sit at room temperature for 10 min.

10. Centrifuge tubes at 10,000 rpm at 4°C for 10 min. Discard supernatant.

11. Wash pellet with 20 ml of 75% ethanol. Vortex briefly.

12. Centrifuge at 10,000 rpm at 4°C for 10 min. Discard supernatant and dry pellet.

13. Add DEPC-Water, to pellet. Resuspend RNA by pipetting up and down a few times.

14. Add RNase inhibitor aseERASE to RNA sample

15. Transfer sample to microcentrifuge tube at room temperature.

16. Spin samples at high speed in microcentrifuge tube for 5 min at room temperature.

17. Transfer RNA solution (supernatant) to a new tube. And store it.

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Other methods of RNA extraction

a) Pine tree Method

b) High – Performance Liquid Chromatography (HPLC)

c) QIAamp MinElute Virus Spin Kit

d) QIAamp DSP Virus Kit

e) MagAttract Virus Mini M48 Kit

f) Oligotex mRNA Mini Kit

g) TurboCapture 96 mRNA Kit

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Gel Electrophoresis

• Gel electrophoresis is a technique used to separate macromolecules (DNA, RNA, Proteins) and their fragments on the base f size and charge. RNA is negatively charged and sugar is backbone.

• Gel electrophoresis involve an electrical field.

• There are two types of gel electrophoresis one is agarose gel electrophoresis while the other is polyacrylamide gel electrophoresis.

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Differences between Agarose and Polyacrylamide Gel AGAROSE GEL POLYACRYLAMIDE GEL

Poured Horizontally Poured Vertically

Separate large molecules more than 1 kb

Separate small molecules less than 1 kb

Non-toxic neurotoxin

Mostly used for DNA and RNA separation Used for DNA, RNA or Protein separation

Staining before pouring the gelStaining dye is Ethidium bromide

Staining after pouring the gel Staining dye is Coomassie blue

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Agarose Gel ElectrophoresisMaterials

• Agarose Solution 1%; 1g agarose boiled in the 100 ml of TE buffer

• Gel Loading Dye (bromophenol blue)

• TBE (stock); in a 1L beaker: 850 ml of dH2O, 20 ml of 0.5M EDTA (pH 8), 54g Tris base stir, add 27.5 g Boric Acid, stir, pour into graduated cylinder and fill up to 1L with dH2O, autoclave 0.5M EDTA (pH 8.0) 46.5 EDTA in 200ml water, 5 g of NaOH pellets to adjust pH to 8.0, EDTA won’t dissolve until pH is 8.0

• Ethidium Bromide

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Procedure 1.Pour the 100 ml of melted agarose into the gel casting tray.

2.Apply combs.

3.Let the agarose solidify for at least 15 minutes. Remove the gel plate, with the gel on it, from the casting tray and place it in the gel box.

4.Add running buffer until the gel is completely submergedThe gel is now ready for use.

5.Use a micropipette and a clean tip to transfer RNA sample to a well.

6.Place the lid on the gel box and fit the power cords over the two electrodes.

7.Normally, you would want to stop the gel when the bromophenol blue (dark blue) dye line is near the bottom edge of the gel. Turn off the power supply and disconnect the leads

8.Carefully remove the gel tray and gel from the gel box.

9.After gel running UV light or UV illuminator used to visualize the bands. Gel documentation is also used which advanced from.

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Polyacrylamide Gel ElectrophoresisMaterials and Reagents

Polyacrylamide gel percentageReagents 3.5% 5.0% 8.0% 12.0% 20.0%

30% acrylamide 11.6 16.6 26.6 40.0 66.6

H2O 76.3 71.3 61.3 47.9 21.3

3% ammonium persulphate 2.1 2.1 2.1 2.1 2.1

10× TBE 10.0 10.0 10.0 10.0 10.0

Total 100.0 100.0 100.0 100.0 100.0

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Procedure 1. Prepare the gel solution with the desired polyacrylamide percentage according to the table.

2. Add TEMED to complete the gel before the acrylamide polymerizes.

3. Apply the combs and make sure that no acrylamide solution is leaking from the gel mold.

4. Allow the acrylamide to polymerize for 30-60 minutes at room temperature.

5. After polymerization complete. remove gels from gel caster and insert gels into vertical gel box. Add running buffer and carefully pull the combs from the polymerized gel.

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• 6. Use a Pasteur pipette or a syringe and mix the RNA samples with the appropriate amount of gel loading buffer. Load the mixture into the wells.

• 7. Connect the electrodes to a power pack, turn on the power, and begin the electrophoresis run.

• 8. Run the gel until the marker dyes have migrated the desired distance. Turn off the electric power, disconnect the leads, and discard the electrophoresis buffer from the reservoirs.

• 9. Detach the glass plates. Lay the glass plates on the bench. Use a spacer or plastic wedge to lift a corner of the upper glass plate. Check that the gel remains attached to the lower (white) plate. Pull the upper plate smoothly away. Remove the spacers.

• 10. Stain gels with SYBR gold or dry gels and expose to film or Phospho-Imager screen.

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Gel Electrophoresis depend upon the following factors

• 1. Molecular Weight

• 2. Concentration of Gel

• 3. Voltage

• 4. Temperature

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