6. RNA ISOLATION FROM PLANT TISSUE
INTRODUCTION
Isolation of good quality and intact RNA is highly essential for cloning of
genes from eukaryotic cells. In order to elucidate the regulatory
properties of a gene, it is necessary to know the structure and amount of
RNA produced. RNA from any cell can be copied into double stranded DNA and
cloned, resulting in the production of a cDNA library specific to the cell
type.
A typical mammalian cell contains 105
μg
of RNA, 80-85% of which is ribosomal RNA (28S, 18S, 5.8S and 5S Species).
Most of the remaining 15-20% consists of a variety of low Mol. Wt. Species (eg.
tRNAs and small nuclear RNAs). By contrast mRNA makes up between 1% and 5%
of the total cellular RNA.
Hurdles in the isolation of total RNA:
1.
The difficulty in RNA isolation is that most ribonucleases are very stable
and active enzymes.
2.
The ribose residues carry hydroxyl groups in both the 2’
and 3’ positions, RNA is chemically much more
reactive than DNA, and is easily cleavable by contaminating RNases.
3.
Many RNases are resistant to prolonged boiling and mild denaturants and are
able to refold quickly when denatured.
4.
RNases do not require divalent cations for activity and thus can’t
be easily inactivated by EDTA or other metalion chelators in buffer
solution.
5.
RNases are released from cells upon lysis and are present on the skin,
constant vigilance is required to prevent contamination of glassware and
benchtop and the generation of RNases in aerosols.
Inhibitor of RNases:-
1.
The most commonly used inhibitor is DEPC (Diethyl pyrocarbonate).
2.
It is a highly reactive alkylating agent used to inactivate RNases.
3.
Prepare all solutions and buffers with RNase free glassware and DEPC
treated water.
4.
All the plastic ware and glassware should be treated with 0.1% DEPC prior to
autoclaving.
5.
Autoclaving may not be sufficient to inactivate RNases. So bake glassware
for 4 hrs. at 300°
C.
6.
Degradation of RNA by ribonuclease is avoided by working quickly and keeping
everything cold.
7.
Handle everything by wearing gloves.
Key to successful purification of intact RNA
Cellular RNases should be inactivated as quickly as possible at the very
first stage in the extraction process. Once the endogenous RNases have been
destroyed the immediate threat to the integrity of the RNA is greatly
reduced and we can proceed to isolation of total RNA.
ISOLATION OF RNA USING GUANIDIUM THIOCYANATE:
Many methods for the isolation of intact RNA from cells use strong
denaturants such as Guanidium thiocyanate to disrupt cells, solubilise their
components, and denature endogenous RNases simultaneously. The use of GTC
in RNA extraction, was mentioned by Ullrich et al (1977), Chirgwin et al
(1979) and Hanetal (1987).
PRINCIPLE
The use of guanidium to lyse cells was originally developed to allow
purification of RNA from cells high in endogenous ribonucleases.. GTC, a
stronger chaotropic agent, contains potent cationic and anionic groups that
form strong hydrogen bonds. It is used in the presence of a reducing agent
to break protein disulphide bonds in the presence of a detergent such as
sarkosyl to disrupt hydrophobic interaction.
Advantages of using GTC:
·
GTC a strong denaturant not only disrupts cells and solubilize their
components and also denatures endogenous RNases simultaneously.
·
The protocol takes the advantage of the observation that RNA can be
separated from DNA and protein by virtue of its greater density. Lysis of
cells occurs with a monophasic solution of GTC and phenol.
·
Under these acidic conditions, most proteins and small fragments of DNA
remain in the organic phase while large fragments of DNA and proteins remain
in the interphase.
·
Fragmentation of DNA during homogenization helps to remove DNA from aqueous
phase.
·
Addition of chloroform generates a second organic phase into which DNA and
proteins are extracted, leaving RNA in the aqueous phase.
·
Recovery of total RNA by precipitating with isopropanol.
·
To remove low mol. wt. RNA the recovered RNA is washed with LiCl.
PROTOCOL:
1.
Grind material (1 gm) using liquid nitrogen
2.
Resuspend the powder in 5 ml of GTC buffer in a 13 ml round bottom screw-cap
Sarstadt tube
3.
Add 0.5 ml 2M sodium acetate pH 4.0 and vortex
4.
Add 5 ml of water saturated phenol (3.9 – 4.2) and vortex.
5.
Add 1 ml of chloroform and vortex
6.
Spin for 10 mins at 10,000 xg
7.
Save upper phase and transfer to new 13 ml tube containing 5 ml of
isopropanol.
8.
Place on ice for 10 mins or O/N at –20°
C to precipitate RNA
9.
Spin for 10 mins at 10,000 xg.
10.
Transfer pellet to a 2 ml microcentrifuge tube by adding 1 ml 4 M LiCl2.
DO NOT VORTEX. The pellet should dislodge easily with the addition of the
LiCL2. Rinse tube with an additional 0.5 ml LiCl2 to
remove any left over pellet and combine all this in the 2 ml tube.
11.
Place all 2 ml tubes in a multiple vortex pad and vortex for 10-15 mins
until pellet is completely resuspended.
12.
Spin in microfuge for 5 mins to pellet RNA
13.
Add 1 ml LiCl2 to pellet and repeat vortex and spin 5 mins
14.
Resuspend pellet in ~ 0.7 mL pellet resuspension solution. Make sure the
entire pellet is resuspended (vortex at least 5 mins).
15.
Sequentially extract the aqueous phase with equal volumes of phenol,
phenol/chloroform and 24:1 chloroform/isoamyl alcohol.
16.
Quantify RNA and store it –70°C.
·
The yield of total RNA depends on the tissue or cell source, but it is
generally in the range of 4-7 ug/mg of starting tissue or 5-10 ug/106
cells. The A260/A280 of the extracted RNA is generally 1.8 – 2.0.
Quantification of RNA:
Samples of RNA are denatured by treatment with formamide and separated by
electrophoresis through agarose gels containing formaldehyde.
Principle:
Formaldehyde forms unstable Schiff bases with the single imino group of
guanine residues. These adducts maintain RNA in denatured state by
preventing intrastrand watson crick base pairing. A 1.4% Agarose gel is
suitable for resolving RNA of 0.5 – 8.0 kb size range. Larger RNAs should be
separated on gels cast with 1.0% or 1.2% agarose.
For 50 ml of 1.4% Formaldehyde agarose gel.:
Add 40.8 ml DEPC water (0.1%)
0.7 gms agarose
Boil the above mixture in a micro oven and cool it to 50°
C. Then add 5 ml 10 x MOPS and 1.5 ml formaldehyde (mixed separately and
kept ready). Now it is mixed swiftly and poured in the mould.
Processing of the RNA samples:
Amount of
RNA 25 μ
l
Formamide
20 μl
10x
mops 7.2
μl
Formaldehyde 7.87
μl
RNA
loading dye 12.0
μl
The above samples with all the components are boiled in boiling water for 15
mins. In the mean time of boiling the gel is kept for pre run in 1xMOPS
buffer at 30V. To each RNA sample, add 1.2
μl
of ethidium bromide (50μg/ml) before loading.
SOLUTIONS:
GTC buffer
4M GTC
25 mM sodium
citrate (pH 7.0)
0.5%
Sarkosyl
14.3 M
betamercapto ethanol
Pellet resuspension solution
0.5% SDS
10 mM Tris-HCl (pH 7.5)
1 mM EDTA
Phenol: Saturated with 0.1% DEPC (pH 4.0) : Melt phenol by keeping at 650
C and add 0.5% of hydroxy quinoline (which helps in distinguishing the
organic layer and also acts as a reducing agent). To the melted phenol add
equal volume of 0.1% DEPC water and keep it on magnetic stirrer for 4-6
hours. Pour into a separating funnel and allow it to remain until a clear
aqueous phase separates. Discard the aqueous layer and collect the phenol
in a beaker. Then add equal volume of 0.1% DEPC water and continue the
extraction process till the pH reaches 4.0. Store it at 40C by
layering with 0.1V of 10.1% DEPC water.
STOCK SOLUTIONS:
·
DEPC water (1000 ml distilled water + 1 ml DEPC ): Keep it for overnight
stirring to make 0.1% DEPC
·
2M Sodium acetate (pH 4.0 ) : mol.
Weight is 82.03. For 100 ml solution add 16.4 gms in 0.1% DEPC water .
Adjust the pH to 4.0 using glacial acetic acid.
·
Sodium citrate 1M (pH 7.0) Mol. Weght 294.1: For 100 ml solution add 29.4
gms in 0.1% DEPC and adjust the pH to 7.0 using HCl.
·
1M Tris (pH 7.5) Mol. Weight 121.14. For 100 ml solution add 12.114 gms in
water and adjust the pH 7.5 using HCl. In aqueous solutions:Depc hydrolysis
rapidly to Co2 and ethanol. This hydrolysis is greatly
accelerated by Tris and other amines, which themselves become consumed in
the process. DEPC can’t
be used to prepare these solutions.
·
4M Lithium
chloride: Mol. Weight 42.39gms : To 100 ml solution, add 16.9 gms of lithium
chloride.
·
0.5 M EDTA
(pH 8.0 using NaoH): Mol. Weight 372.24 : For 100 ml solution add 17.6 gms
of EDTA in 0.1% DEPC and keep it for overnight stirring. Keep adding NaOH
pellets until EDTA gets completely dissolved. (EDTA dissolves only at pH
8.0).
·
10xMOPS:
0.2M
MOPS
20mM
Sodium acetate
10mM
EDTA (pH 8.0)
First
dissolve MOPS in 0.1% DEPC water. Adjust to pH 7.0 with 2N NaOH. Then add
sodium acetate and EDTA at 20 and 10 mM concentration respectively. Filters
sterilize it.
NOTE: All the above stock
solutions should be autoclaved and handle them only with gloves.
|
