3. 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
, tRNA and rRNA
3
4. INTRODUCTION
Ribonucleic acid can be isolated from plant tissue for the
purpose of:
– mRNA isolation
– In vitro translation
– Northern analysis
– cDNA library construction
Rigorous ribonuclease free environment is to be
maintained
All glasswares, plasticwares and reagents made RNAse
free (using 0.01% DEPC)
Next day, DEPC is inactivated by autoclaving for 30 min
4
6. 6
• 4M Guanidine thiocyanate, 42 mM
sodium citrate, 0.83% N- lauryl
sarcosine and 0.2 mM 2-
mercaptoethanol
Denaturing
buffer
• Saturated with 50mM sodium
acetate till the pH of upper aqueous
phase is approximately 4.0
Phenol
• Acid saturated phenol: chloroform:
isoamyl alcohol(25:24:1)
Phenol-
chloroform
CONTINUE………
7. 7
Collect, wash, blot dry and weigh fresh tissues
Grind tissue to fine powder under liquid nitrogen and
extract with 4ml/gm pre-chilled denaturing buffer
Transfer the homogenate to centrifuge tubes
Add sodium acetate and centrifuge the slurry for 20 minutes
Transfer supernatant to other tube and add phenol
chloroform
PROCEDURE
Mix thoroughly and chill on ice for 15 minutes
8. 8
Separate the phases by centrifugation
Remove the top aqueous phase containing RNA carefully to
fresh tubes
Add isopropanol and allow RNA to precipitate overnight
Next day, RNA is pelleted by centrifugation at 10,000 rpm
Resuspend the RNA pellet and heat briefly upto 65⁰C
Reprecipitate and pellet RNA with isopropanol
Resuspend the pellet in 75% ethanol and centrifuge it
Pellet is dried briefly under vacuum , resuspend in RNAse
free water and stored at -70⁰C
9. PRECAUTION
A separate corner should be in lab for RNA work
which is kept free from RNAse contaminant
Phenol have to be acid saturated to a pH nearing 4.0
High concentration of Guanidine thiocyanate should
be used
Before removing Guanidine thiocyanate, all the
protein should be removed
Disposable gloves should be used and changed
frequently
9
10. Extraction of RNA
CTAB 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
• Before use add 1% beta marcaptoethanol.
10
12. 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 55-65 °C 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 impurities settled down at the
bottom after centrifugation and upper liquid is formed
which is called supernatant.
• Supernatant is shifted to another test tube and mixed with
equal amount of chloroform : Isoamyl alcohol.
• Then sample is vortex for 3-5 minutes then centrifuged at
maximum speed for 10 minutes.
12
13. • The pellet settles down at bottom and
supernatant is shifted to another Eppendorf.
• Added equal amount of Iso-propanol and
incubated at -20 °C for 5 minutes.
• Then is again centrifuged at maximum speed
RNA settle down at the bottom.
• Then it is washed with 70% ethanol and
centrifuged again pellet down at the bottom.
• 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.
13
14. • 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
14
15. TRIzol Method
• This is a modification of the procedure originally
described by Chomczynski P. and Sacchi N.
1987.
• The correct name of the method is guanidinium
thiocyanatephenol- chloroform extraction.
• TRIzol is light sensitive and is often stored in a
dark-colored, glass container covered in foil. It
must be kept below room temperature.
• When used, it resembles cough syrup, bright
pink. The smell of the phenol is extremely
strong.
15
16. Material and Reagents
• 1) TRIzol Reagent (Commercially available
from many venders)
• 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%
16
17. • 0.8 M sodium citrate / 1.2 M NaClO
• Isopropanol (2-Propanol)
• Chloroform
• DEPC-Water
• 75% ethanol prepared with DEPC-Water
• RNase Inhibitor (e.g., aseERASE TM BIO 101
Cat. 2601-104)
• 50 mL sterile plastic screw-cap centrifuge tubes
17
18. PROCEDURE
• Grind 1g tissue in liquid nitrogen in a mortar and
pestle.
• Transfer powdered tissue to a sterile plastic screw-cap
centrifuge tube containing TRIzol reagent.
• Incubate samples at room temperature for 5 min.
• Homogenize tissue with homogenizer for 15 seconds.
• Centrifuge samples at 12000 rpm at 4°C for 10 min.
• Transfer supernatant into new sterile plastic screw-cap
centrifuge tube, Discard pellet.
• Add chloroform tube and shake it vigorously with
vortex for 15 sec.
– Let tube placed at room temp 2-3 min. Centrifuge tube at
10,000 rpm at 4°C for 15 min.
– Carefully pippet aqueous phase into a clean screw-cap
centrifuge tube, discard interphase and lower phase into
waste.
18
19. • 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.
• Centrifuge tubes at 10,000 rpm at 4°C for 10 min.
Discard supernatant.
• Wash pellet with 20 ml of 75% ethanol. Vortex
briefly.
• Centrifuge at 10,000 rpm at 4°C for 10 min. Discard
supernatant and dry pellet.
• Add DEPC-Water, to pellet. Resuspend RNA by
pipetting up and down a few times.
• Add RNase inhibitor aseERASE to RNA sample
• Transfer sample to microcentrifuge tube at room
temperature.
• Spin samples at high speed in microcentrifuge tube
for 5 min at room temperature.
• Transfer RNA solution (supernatant) to a new tube.
And store it.
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20. MORE TECHNIQUES (RNA ISOLATION)
Organic Extraction Methods
• Organic extraction methods are considered the
gold standard for RNA preparation.
• During this process, the sample is homogenized
in a phenol containing solution and the sample
is then centrifuged.
• During centrifugation, the sample separates into
three phases: a lower organic phase, a middle
phase that contains denatured proteins and
DNA, and an upper aqueous phase that contains
RNA.
• The upper aqueous phase is recovered and RNA
is collected by alcohol precipitation.
20
21. Benefits of organic extraction
• Rapid denaturation of nucleases and
stabilization of RNA
Drawbacks of organic extraction
• Laborious and manually intensive processing
• Difficult method.
21
22. Filter-based RNA isolation
• Filter-based, spin basket formats utilize
membranes that are seated at the bottom of a
small plastic basket.
• Samples are lysed in a buffer that contains
RNase inhibitors (usually guanidine salts), are
bound to the membrane by passing the lysate
through the membrane using centrifugal force.
• Wash solutions are passed through the
membrane and discarded.
• An appropriate elution solution is applied and
the sample is collected into a tube by
centrifugation.
22
24. Benefits of spin basket formats
• Convenience and ease of use
• Ability to isolate RNA and DNA.
• Ability to manufacture membranes of various
dimensions
Drawbacks of spin basket formats
• Propensity to clog with particulate material
• Retention of large nucleic acids such as gDNA
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25. Magnetic Particle Methods
• Magnetic particle methods utilize small (0.5–1
μm) particles that contain a paramagnetic core.
• Paramagnetic particles migrate when exposed to
a magnetic field, but retain minimal magnetic
memory once the field is removed.
• This allows the particles to interact with
molecules of interest based on their surface
modifications, be collected rapidly using an
external magnetic field, and then be
resuspended easily once the field is removed.
• Samples are lysed in a solution containing
RNase inhibitors and allowed to bind to
magnetic particles.
• The magnetic particles and associated cargo are
collected by applying a magnetic field.
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