2. RNA extraction
RNA extraction is the purification of RNA from
biological samples. This procedure is complicated by
the presence of ribonuclease enzymes in cells and
tissues.
3. Isolation of RNA
⢠Requires STRICT precautions to avoid sample
degradation.
⢠RNA especially labile.
4. RNAses
⢠RNases are naturally occurring enzymes that degrade
RNA
⢠Common laboratory contaminant (from bacterial and
human sources)
⢠Also released from cellular compartments during
isolation of RNA from biological samples
⢠Can be difficult to inactivate
5. RNAses
⢠RNAses are enzymes which are small proteins that can
renature and become active.
⢠MUST be eliminated or inactivated BEFORE isolation.
⢠CRITICAL to have a separate RNAse free area of lab.
6. Protecting Against RNAse
⢠Wear gloves at all times
⢠Use RNase-free tubes and pipet tips
⢠Use dedicated, RNase-free, chemicals
⢠Pre-treat materials with extended heat (180 C for several
hours), wash with DEPC-treated water, NaOH or H2O2
⢠Supplement reactions with RNase inhibitors
7. TRIZOL RNA Isolation Protocol
TRIZOLE REAGENT
The correct name of the method is guanidinium thiocyanate-
phenol-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..
Caution should be taken while using TRIzol (due to
the phenol and chloroform).
Exposure to TRIzol can be a serious health hazard. Exposure can lead to
serious chemical burns and permanent scarring
. A lab coat, gloves and a plastic apron are recommended
8. PRINCIPLE
⢠TRIzol Reagent is a ready-to-use reagent used for RNA isolation from cells
and tissues.
⢠TRIzol works by maintaining RNA integrity during tissue homogenization,
while at the same time disrupting and breaking down cells and cell
components.
⢠Addition of chloroform, after the centrifugation,
separates the solution into aqueous and organic phases.
⢠RNA remains only in the aqueous phase.
⢠After transferring the aqueous phase, RNA can be recovered by
precipitation with isopropyl alcohol. But the DNA and proteins can
recover by sequential separation after the removal of aqueous phase.
⢠Precipitation with ethanol requires DNA from the interphase, and an
additional precipitation with isopropyl alcohol requires proteins from the
organic phase. Total RNA extracted by TRIzol Reagent is free from the
contamination of protein and DNA.
⢠This RNA can be used in Northern blot analysis, rt-pcr, in vitro
translation, RNase protection assay, and molecular cloning
10. Homogenization:
1. Tissues: Homogenize tissue samples in 1 ml of
TRIZOL reagent per 50 to 100 mg of tissue using
a glass-Teflon or power homogenizer.
2. Cells grown in Monolayer: Rinse cell monolayer
with ice cold PBS once. Lyse cells directly in a
culture dish by adding 1 ml of TRIZOL Reagent
per 3.5 cm diameter dish and scraping with cell
scraper. Pass the cell lysate several times
through a pipette. Vortex thoroughly.
11. 2. Phase Separation
â˘The homogenized samples were incubated for 5 minutes at 15
to 30°C for the complete dissociation of nucleoprotein
complexes.
â˘0.2 ml (200 microliters)of chloroform per 0.75 ml of TRIZOL
LS Reagent was added. The tubes were shaked vigorously by
hand for 15 seconds and incubated them at 15 to 30°C for 2
minutes.
â˘The samples were centrifuged for 15 minutes at no more than
12,000 g (4°C).
â˘The aqueous phase was transferred to other tubes. ( Following
centrifugation, the mixture separates into a lower red, phenol-
chloroform phase, an interphase, and a colorless upper
aqueous phase. RNA remains only in the aqueous phase. The
volume of the aqueous phase is about 70% of the volume of
TRIZOL Reagent used for homogenization.)
13. 3. RNA Precipitation
â˘The RNA was precipitated from the aqueous phase
by mixing with 3 microlitre of glycogen and 500
microlitre of isopropyl alcohol.
â˘The mixture was centrifuged for 30 minutes at
12,000 à g (2 to 8°C).( The RNA precipitate forms
a gel-like pellet on the side of the tube at bottom).
14.
15. 4. RNA Wash
1.The supernatant was removed. The RNA pellet was
washed once with 75% ethanol, adding 900 microlitre
of 75% ethanol per 0.75 ml of TRIZOL Reagent used
for the initial homogenization.
2.The sample were inverted and mixed and
centrifuged at 12,000 rpm for 30 minutes at 4
degree.
16. 5. Redissolving RNA
â˘The RNA pellet was dried .
â˘RNA was dissolved in RNase-free water (or 0.5%
SDS solution) by passing the solution through the
pipette tip for a few times, and incubating for 10
minutes at 55 to 60°C.
17.
18. 6. SPECTROPHOTOMETRIC ANALYSIS:
⢠Dilute 1 Οl of RNA with 39 Οl of DEPC-treated water (1:40
dilution).
⢠Using 10 Οl microcuvette, take OD at 260 nm and 280 nm to
determine sample concentration and purity.
⢠The A260/A280 ratio should be above 1.6.
⢠Apply the convention that 1 OD at 260 equals 40 ¾g /ml RNA.
Example: Use the buffer in which the RNA is diluted to zero the
spectrophotometer:
â˘Volume of RNA sample = 100 Âľl
â˘Dilution = 10 Âľl of RNA sample + 390 Âľl distilled water (1/40 dilution)
â˘Absorbance of diluted sample measured in a cuvette (RNase-free):
A260 = 0.23
â˘Concentration of original RNA sample = 40 x A260 x dilution factor =
40 x 0.23 x 40
â˘RNA concentration: 368 Âľg/ml 0r 368 ng/ul
19. Other Methods of RNA ISOLATION
Filter-based RNA isolation
Magnetic Particle Methods
20. 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. Filter-based RNA isolation
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
23. Magnetic Particle Methods
Magnetic particle methods utilize small (0.5â1 Âľm) 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.
