BSL509- Plant Molecular and Genetic

Engineering

Assignment on

Isolation and purification of RNA

Submitted to :- Submitted by

Prof. Avinash kaur Nagpal Alisha Kumari

M.Sc. Bot (sem-3)

2017BSB0101

Botanical and environmental science Department.

GNDU,amritsar
Overview of RNA:-
RNA:-Ribonucleic acid is the major second nucleic acid in cell serves as a genetic messenger
passing the information stored in DNA to other parts of cell for protein synthesis.it is single
strandard structure . The unassembled monomers of both RNA and DNA are called nucleotides.
These building blocks consist of three key component:-a pentose sugar, a phosphate group and
nitrogeneous base .A nitrogenous base joined to the pentose sugar and form nucleoside

Base + sugar = nucleoside

Nucleoside + phosphate = nucleotide

The key chemical difference between the RNA and DNA is the presence of five-carbon sugar
ribose, in which a hydroxyl group (-OH) is joined to the 2` carbon of the ribose sugar whereas
the absence of this –OH group in the DNA is the underlying basis on the name of sugar
deoxyribose .In addition ,one finds the base uracil in RNA , substituted in the DNA by closely
pyrimidine thymine ,though it is possible to find nucleotides containing uracil in certain situation

Fig 1:- Structure of Purines and Pyrimidines

 Fig 2:- structure of RNA

The major forms of RNA exist in every cell:-

Messenger RNA :- the process of transcription results in synthesis of the mRNA from DNA. The
mRNA.carriers genetic information of DNA from nucleus to cytoplasm.it can be monocistronic
as in eukaryotes or polycistronic in prokaryotes.

Fig 3:-Structure of mRNA

Transfer RNA:- At the time of protein synthesis,these RNA recognize the coded genetic
information of mRNA and bring specific amino acid at the site of growing polypeptide
chain.There is atleast one tRNA for each amino acid.

Ribosomal RNA:- it is most abundant form of RNA and counts about 80% of total cellular
content . it is synthesized in nucleolus .In cytoplasm, ribosomal RNA and protein combine to
form ribosomes.
Why isolate RNA?
mRNA synthesis is a dynamic expression of genome of an organism. As such, mRNA is central
to information flow within a cell. The isolation of RNA with the high quality is crucial step
required to perform various molecular biology experiment. TRIzoL reagent is used for RNA
isolation from cell to tissue.

Goals in purification of RNA:-

Goal 1:- Select an appropriate method of membrane solubilization :- the first decision to factor
into design of an RNA isolation strategy is the method of cell disruption, and it is based on
which population of RNA or subcellular compartment the investor wishes to study.

Goal 2:- Ensure total inhibition of nuclease activity :- The imperative for controlling nuclease
acvtivity should be abundantly clear. This include purging RNase from reagents, glasswares
,consumable plastic ware, and equipment and controlling such activity in cell lysate. Steps for
the inhibition or elimination of RNase activity must, first and foremost, demonstrate
compatibility with the lysis buffer

Goal 3:-Select a method for deprotienization of the sample :- The complete removal of protein
from cellular lysate is important in isolation of both DNA and RNA. Any procedure for the
deprotienization itself a mean of controlling RNase activity.

Goal 4:- Select a method for nucleic acid concentration :- This is final step in most RNA
purification schemes. The most versatile method for concentrating solution nucleic acids in
precipitation using various combination of salt and alcohol.
The method for isolation and purification of
RNA as follows:-
1.Organic extraction method

2.Filter-based, spin basket format

3.Magnetic particle methods

4. Direct lysis method.

1 .Organic extraction method :- This method involve phase separation by addition and
centrifugation of mixture of a solution containing phenol and chloroform and a chaotropic agent
(Guanidinium thiocyanate) and aqueous sample. Guanidinium thiocyanate result in denaturation
of proteins and RNase ,seprating rRNA from ribosomes. Addition of chloroform forms a
colourless upper aqueous phase containing RNA , an interphase containing DNA and lower
phenol-chloroform phase containing protiens. RNA is collected from upper aqueous phase by
alcohol precipitation followed by rehydration.

Guanidinium-acid-phenol extraction technique

The highest quality RNA indisputably results from the extraction of RNA mediated by
chaotropic lysis buffers, and guanidinium –containing buffers are among the most effective.
These isolation procedures have the advantage of disrupting cells grown in culture or whole
tissue samples rapidly and completely ,simultaneously inactivating RNase activity, even when it
is present in great abundance. The RNA is isolated in very short term by treatment of cells with
guanidinium thiocyanate or tissue lysate with an acidic phenol solution; chloroform is added to
facilitate partitioning of the aqueous and organic materials. Although such extraction buffers are
easily prepared in the molecular biology laboratory.
2 .Filter- based , spin basket formats:- This method utilize membranes(usually glass fiber,
derivitized silica, or ion exchange membranes) that are seated at the bottom of a small plastic
basket. Sample are lysed in a buffer that contain RNase inhibitors(usually guanidinium salts),
and nucleic acid are bound to the membrane by passing the lysate through the membrane using
centrifugal force. Wash solution are subsequently passed through membrane and discarded. An
appropriate elution solution is applied and the sample is collected into the tube by centrifugation.
Some formats can be processed by either centrifugation or vaccum using specialized manifolds.
Hybrid methods that combine the effectiveness of organic extraction with the ease of sample
collection ,washing, and elution of spin basket formats also exist.

BENEFITS:-

*Convenience and ease of use.

*Amenable to single-sample and 96-well processing.

*Ability to automate.

*Ability to manufacture membranes of various dimensions.

DRAWBACKS:-

*Propensity to clog with particular material.

*Retention of large nucleic acid such as gDNA.

*Fixed binding capacity within a manufactured format.

*When automated ,requirement for complex vaccum systems or centrifugation.

3 .Magnetic bead method:- This is simple and efficient way which is used in purification of
nucleic acid nowadays . Many magnetic carriers are now commercially available .Particles
having magnetic charge may be removed in application of magnetic field.

A magnet can be applied to the sides of vessels which contains the sample mixtures for the
aggregating the particles near the wall of vessels and pouring away the remainder of samples.

Particles having the magnetic or paramagnetic properties are employed in an invention where
they are encapsulated in a polymer such as magnetizable cellulose. In the presence of certain
concentrations of salt and polyalkylene glycol, magnetizable cellulose can bind to nucleic acid.
Small nucleic acid required higher salt concentrations for strong binding to magnetizable
cellulose particles. Therefore salt concentrations can be selectively manipulated to release
nucleic acid bound to magnetizable cellulose on the basis of size .The magnetizable cellulose
which bound with nucleic acid will be washed with suitable wash buffer before they are
contacted with suitable elution buffer to separate out the desired nucleic acid with cellulose.
Separation of magnetizable cellulose from supernatant during all the purification steps can be be
done by applying magnetic field to draw down or draw them to the side of vessel. The magnetic
component of cellulose can also be substituted by other magnetic compounds such as ferreous
oxide or nickel oxide.

Magnetic oligo(dT) bead is an alternative to other oligio (dT) matrices for the purification of
poly(A)+ RNA from total RNA sample. The poly(A)+RNA can be extracted by introducing
magnetic beads coated with oligio(dT).RNA with a poly-A tail attach to the oligio(dT).The beads
will then be drawn to the bottom of a tube removing mRNA directly from the total RNA.The
magnetic beads which are specially treated minimize the nonspecific binding of other nucleic
acid and ensure the purity of mRNA.
4 .Direct lysis method:- This method involve uses of lysis buffer under the specified conditions
for the disruption of sample and staiblisation of nucleic acid. If desired, sample can be purified
from staibilised lysates .This method eliminates need of binding and binding of elution from
solid surface and thus avoid bias and recovery effeciancy effect.

Gentle lysis buffers:- cellular lysis mediated by non- ionic, hypotonic buffer is not disruptive to
most subcellular organelles. The inclusions of non- ionic detergents such as NP-40 and MgCL in
the lysis buffer facilitates plasma membrane solubilization while maintaining nuclear integrity.

In this protocol, intact nuclei, large organelles, and cellular debris are easily removed by
differential centrifugation. The resulting cytosolic supernatant is rich in cytoplasmic RNA and
protiens, the latter being easily removed by series of extractions with the mixture of organic
solvents such as phenol and chloroform or by silica column chromatography .The advantage of
this method is that the RNA is precipitate at the end of the procedure represent only the
cytoplasmic population

The disadvantage of this approach is that the lysis buffer alone is not sufficient chaotropic to
fully inhibit RNase activity. Keep in mind that upon cellular lysis, normally sequestered RNases
are suddenly liberated, and their activity will greatly compromise the integrity of the RNA even
as investigator is working diligently to purify it

This approach is particularly suited for the isolation of the nuclear RNA.

*Analysis of RNA :-

Most important and certainly the most often used technique in RNA analysis is Gel
Electrophoresis. This technique is generally applicable for RNA detection, Quantification,
purification by size. RNA are negatively charged, they migrate toward the anode in presence of
electric current. The gels acts as a sieve to selectively impede the migration of RNA in
proportion to its mass, given that its mass is generally proportion to its charge

The various methods of gel electrophoresis is:-

1. Polyacrylamide gel electrophoresis

2. Agrose gel electrophoresis.
*Determining the yield and quality of purified RNA :-

1. Determining yield by spectroscopy or fluorometry :- The easiest way to determine the

quantity of RNA in a sample is to measure the absorbance at 260 nm using a
spectrophotometer. Because the bases in RNA absorb UV light in 250-265 nm range.

Procedure :- This procedure is simple. Place 1.0 -1.5 microlitre of the RNA onto the sample
pedestal. The UV absorbance of the sample is then read either at fixed wavelength or in a UV
visible scan. Pure RNA sample are read at 260 nm or 280 nm. It is possible also to use these
instruments to scan a full UV and visible wavelength absorbance spectrum, from 250 nm to 750
nm. The UV-visble wavelength scanning procedure is more useful in microarray studies when
one wants to quantitate flourochrome dye coupling to cDNA

*Fluorescent dye binding for RNA and DNA quantitation :- RNA can be quantified using
fluorescent dye binding. This is a sensitive assay for detecting and determining the quantity of
RNA present in purified RNA sample. It is 1000 times more sensitive than using UV absorbance
and can detect RNA at 1 ng/mL. The ribogreen RNA reagent, a proprietary fluorescent dye,
preferentially binds to RNA, but it can also detect DNA. This method is useful when making
RNA from nuclear fraction that might be contaminated with the DNA and for assaying very
small quantities of RNA prepared from limited quantities of starting material.

*Determining yield by gel electrophoresis:- For a sample of total or cytoplasmic RNA, a simple
or straight forward way to determine yield is to separate a small aliquot of the RNA on an agrose
gel and stain with the ethidium bromide or SYBR gold. Bands of the rRNA are visualized, and
their intensity is compared to that of a preparation of known quantity

An advantage of this method is that it measured both the quantity and quality of RNA; i.e., sharp
and distinct rRNA bands without a pronounced haze below them is a good sign that preparation
is not significantly degraded. The only disadvantage of this approach is sensitivity; at least 500
mg of RNA must be available to sacrifice for this assessment. To assess quality,sufficient
quantity must be loaded to detect degraded materials.

By far, northern blotting is best method for assessing the quality of the RNA because this
technique visualize the entire RNA, it is diagnostic for any degradation
*Purification of RNA by using TRIzol:- TRIzol solubilization and extraction is a relatively
recently developed second general method for deprotienizing RNA. This method is particularly
advantageous in situations where cell or tissues are enriched for endogenous RNase or when
separation of cytoplasmic RNA from nuclear RNA is impractical

TRIzol is monophasic solution of phenol and guanidinium isothiocyanate that simultaneously

solubilize biological materials and denatures protiens. After solubilization, tha addition of the
chloroform causes phase separation like PCA extraction, where proteins is extracted to the
organic phase, DNA goes to the interface, and RNA remains in aqueous phase. Therefore, RNA
or DNA , can be purified from the same sample. TRIzol extraction is an effective method for
isolating small RNAs, such as microRNAs, piRNAs, or endogeneous, small interfering RNAs.
References:-

1.Keith Wilson and John walker.Principles and techniques of

biochemistry and molecular biology,7th edition. Cambridge
university press,New york.

2.Journal of biomedicines and biotechnology. Published by

hindwai publishing corporation. Article ID:-574398.

3.Robert E. Farrell. RNA methodologies , a labortary guide for

isolation and characterization,4th edition Published by Elsevier
publications.

4. http://nptel.ac.in.>courses>module4

5.http://www.thermofisher.com>references.