methods of isolation and extraction of RNA by using different source such as plant tissues, bacterial culture, etc. 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
Total RNA is isolated and separated from DNA and protein after extraction with a solution called as Trizol. Trizol is an acidic solution containing guanidinium thiocyanate (GITC), phenol and chloroform. GITC irreversibly denatures proteins and RNases. This is followed by centrifugation.
3. 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
5. Take plant material (bean seed/ cauliflower florets)and cut
into small pieces.
Add the extraction buffer and make the fine paste
Transfer the homogenate to flask add SDS and mix it then
incubate at 650C for 15 min in water bath
Add potassium acetate solution mix it and incubate for
30min
Centrifuge the content at 20000rpm for 15 min.
Take supernatant in tubes and add sodium acetate and
95% of ethanol.
place the tube in ice bath for 15min for precipitate RNA
Then centrifuge the tube on 20000rpm for 15min and
discard supernatant
And pellet wash with ice cold ethanol and dry it.
7. Take cell suspension culture in centrifuge tube and
centrifuge at 3000rpm for 5 min.
Re suspend collected cells in PBS , centrifuge it and
discard supernatant
Re-suspended cell in lysis buffer and incubate on ice bath
for 5 min.
Centrifuge tube for 2min at 40C and transfer supernatant
in new tube and add SDS then vortex it.
Add proteinase K and incubate at 370C for 15min.
Add phenol-chloroform-isoamyl alcohol and vortex it for
1min and centrifuge the tube.
Transfer upper layer in new tube and repeat above step 2
times.
New supernatant add sodium acetate and ethanol ,mix it
well and incubate on ice bath for 15-30 min for precipitate.
8. Isolation of total and polymer RNA
Requirements :
• RNA extraction buffer: Lithium chloride, LiCi, SDS, Tris-
NaOH, EDTA
•Lithium chloride( LiCi) 8M: dissolve 34 g of LiCi in 100ml
of water and autoclave store in freeze.
•Distilled phenol with 0.1% hydroxyquinoline , chloroform ,
ethanol .
•Diethylpyrocarbonate water (DEPC)
9. • 1:1mixture of RNA extraction buffer and phenol with hydroxyquinoline and heat it
to 90oCin water bath.
• take plant material and add liquid nitrogen and grind it fine homogenous powder.
• Homogenate to clean flask on ice bath and phenol extraction buffer and gently mix.
• Transfer flask in water bath ,incubate sample at 90oC then it forms milky
suspension.
• Suspension cool on room temp. and centrifuge it at 300rpm for 5min
• Then add chloroform and incubate shaking incubator for 30min.
•Centrifuge these suspension at 20000rpm for 30 min .
•Remove aqueous phase and transfer it into new flask add chloroform.
•Shake the content at 3000rpmfor 15 min.centrifuge content at 12000rpm for 15 min.
•Remove upper phase and transfer it into new centrifuge tube add LiCi ,mix it and
precipitate the RNA for 16-48 hr at 4oc
•Centrifuge precipitate at 4oC on 12000rpm for 30min and wash pellet with LiCi and
twice with ethanol.
•Dry pellet and suspended it in DEPC and store in freeze
10. Extraction of polysomes and polysomal RNA
Material :
Polysome buffer : Nonidet P40, MgCl, EGTA, Tris-NaOH,
NaCl.
Gradient buffer : MgCl2, EGTA, Tris-NaOH, NACl.
GB+ EDTA:75% W/V sucrose in GB: sucrose, DEP
RNA extraction buffer: LiCl, phenol, chloroform, ethanol.
11. Harvest the plant material and grind in liquid Nitrogen
Transfer powder add plant material in ice-cold polysome buffer and
form the slurry of these material
This mixture/slurry cenrifuge at 27000rpm for 10min at 00C. Filter the
supernatant solution.
Filtrate into Beckmann polycarbonate tube with sucrose in GB .
Centrifuge this for 3hr at 40000rpm then place the tube inverted on
sterile paper towel.
dry pellet re-suspended in frozen liquid nitrogen and store at -800C
Carefully load upto 5 mg polysome on gradient and centrifuge for
30min at 40000rpm
Polysome containing fraction are collected and precipitate overnight
in 96% ethanol.
These precipitate centrifuge at 20000rpm for 30min
Extraction of RNA from ethanol precipitate polysome by
phenol/chlorofrom/ LiCl method.
13. Collect the bacterial culture at log phage
and centrifuge it at 5000rpm for 5 min.
Resuspend the pellet in lysis buffer and
add VRC
Add buffered phenol, vortex it for 1 min.
add chloroform and vortex it 1 min.
Centrifuge the content for 10 min at
10000rpm and collect aqueous phase in
new tube
Add sodium acetate and 100% ethanol
mix it well and centrifuge it for 10 min at
10000rpm then re suspend pellet in VRC
Extract pellet with phenol:chlorofrom and
re precipitate the RNA same as above step
and dry the pellet and dissolve it in sterile
D/W and store at -70oC
