Molecular Biology Labs 11
Extraction of RNA from Cultured Cells
Background:
Cells have multiple types of RNA molecules. Most (85%) is in the form of ribosomal
RNA including a 28S, 18S, and 5S species. Most of the remainder (14%) consists of
small specialty RNAs including transfer RNA, and small nuclear RNAs. The really
interesting RNA population is the messenger RNA (mRNA), also called poly A+ RNA,
because these molecules have long poly A tails at their 3’ end. Although the mRNA
encodes all of the proteins in any cell, it accounts for only 0.5- 1% of all cellular RNA.
Thus, most of the RNA molecules isolated in a typical extraction of cultured cells are not
of particular interest to molecular biologists who are usually studying gene expression. In
fact, the presence of large amounts of ribosomal and other RNAs often complicates
analysis of gene expression.
It is possible to purify mRNA from this stew of different RNAs by using a technique
called oligo-(dT) cellulose chromatography. This process is based on the fact that almost
all eukaryotic mRNA molecules have a poly A tail. This mRNA can be separated from
the bulk of nonpolyadenylated RNA by affinity chromatography by binding to the
repeating tracts of thymidine attached to cellulose (oligo-dT cellulose). Once the polyA
RNA is bound, the other RNAs are eluted from the column. Then the mRNA is eluted
from the oligo-dT cellulose by washing with a low salt buffer to destabilize the A-T
interactions. About 1% of the input RNA is recovered as polyA+ RNA. This fraction
works extremely well for analysis of gene expression (reverse transcriptase-PCR,
Northern blotting, and cDNA libraries).
The most difficult problem in isolating good quality RNA is that RNA is not as
chemically stable as DNA. In particular, the enzyme that naturally degrades RNA
molecules within cells, RNase, is ubiquitous. It is present everywhere. It’s all over your
skin, on your fingers, hair, doorknobs, countertops, etc. These RNases are extremely
difficult to inactivate. They resist boiling and they do not need divalent cations for
optimal activity as do DNAses. The bottom line is that it is very difficult to work with
RNA! There are some precautions that one should follow when isolating RNA. These
include:
1. wear gloves and change them frequently when you touch skin or other
contaminated surfaces
2. keep all of your reagents used for RNA extractions separate from your other
reagents
3. use diethylpyrocarbonate and other inhibitors of RNase activity to treat your glass
and plastic ware.
4. bake or autoclave all pipette tips, eppendorf tubes, and glassware that will come
in contact with the RNA.
There are several inhibitors of RNase activity. Diethylpyrocarbonate (DEPC) is a highly
reactive alkylating agent that destroys activity of RNases. Typically, DEPC is added to
water at 0.1% and used to pretreat plastic and glass surfaces. Vanadyl ribonucleoside
complexes are a second inhibitor. They bind to RNase and completely inhibit its activity.
However, the action is reversible, and the inhibitor must be present in all solutions to be
effective. A third class of inhibitors are the naturally occurring proteins that block RNase
activity. They have a remarkable affinity for RNase ( 1-70 femto molar binding affinity).
They can be purchased from several biotech companies (i.e., RNasin).
There are 2 major methods for purifying RNA from cultured cells. Both utilize
guanidinium salts, chaotropic agents that denature proteins. Guanidinium isothiocyanate
is commonly used in combination with sodium sarkosyl (detergent). Both help to break
open the cells and the guanidinium isothiocyanate also inactivates any RNases. The first
method (Chirgwin method) involves layering the cell lysate on a cesium chloride
gradient. The gradient is spun in an ultracentrifuge to separate the macromolecules. The
RNA has the lowest buoyant density and rapidly pellets on the bottom of the tube. The
proteins and DNA remain in the gradient. Although this method is not very labor
intensive, it takes 24 hours for the gradient to work. In addition, it is difficult to process a
lot of samples in the ultracentrifuge. A second method (Chomczynski method) has the
advantage of being fast and able to process many samples at once. In this method phenol
is added to the cell lysate containing guanidinium isothiocyanate and this mixture is
extracted with chloroform. The chloroform allows the mixture to form 2 distinct phases.
The RNA remains in the aqueous phase while the DNA and protein move to the organic
phase. The aqueous phase is removed and the RNA is precipitated by addition of
isopropanol. The RNA is then collected by centrifugation. The entire procedure takes less
that 3 hours and yields high quality RNA. An added advantage of this procedure is that it
allows one to simultaneously purify protein and DNA from the same sample.
Once the RNA sample has been precipitated from the solution containing guanidinium
isothiocyanate, it is again highly susceptible to degradation by RNases. There are 3
methods for storing the purified RNA:
1. Dissolve the RNA pellet in deionized formamide and store at -20C. The
formamide provides a chemically stable environment.
2. Dissolve the RNA in sterile water and store at -80C. This method is not as good.
3. Precipitate the RNA with salt and ethanol and store at -80C. The RNA is very
stable under these conditions.
The RNA can be fractionated on agarose gels containing formaldehyde. Formaldehyde is
a carcinogen and is very toxic. Handle with care and avoid contact with skin or inhalation
of fumes. The formaldehyde is used to maintain the RNA in a denatured state during
electrophoresis. It is incorporated into the sample before loading the gel. It is also
included in the gel and can be added to the running buffer. About 10-20 g of RNA are
loaded into each well. The RNA is heated in the presence of formaldehyde and
formamide to denature (remove any secondary structure). No molecular weight markers
are needed for RNA gels because the rRNA is present in massive amounts. This forms 2
dense bands at 28S and 18S. Almost all cell mRNAs are within this size range. RNA is
analyzed by Northern blot hybridization. The Northern blot is essentially the same
procedure as the Southern blot, except that it examines RNA rather than DNA.
Objectives:
The first objective of this lab is to provide background information and practical
instruction in techniques for isolation of total cell RNA from cultured cells.
The second objective is to fractionate the purified RNA on agarose gels containing
formaldehyde to check for quality.
Materials:
Trizol (a proprietary mix of guanidinium isothiocyanate and phenol, cat# 15596-018,
Invitrogen Corp.)
Cell cultures for extraction
PBS for rinsing
Plastic cell scrapers
Sterile polypropylene centrifuge tubes with caps
Syringes and 25 gauge needles
Chloroform
Isopropanol
10X MOPS buffer
formaldehyde (37%)
Centrifuge and JA-20 rotor with rubber inserts
3M sodium acetate
100% ethanol
micropipetters
sterile eppendorf tubes, yellow tips, and blue tips, eppendorf racks
sterile deionized water
1.4% agarose in MOPS buffer with formaldehyde
mingle apparatus, combs, and power packs
gel loading buffer (5 parts glycerol, 2 parts bromophenol blue)
sample suspension buffer (1 part formaldehyde, 1 part 10x MOPS, 5 parts formamide)
65C water bath
0.5% ethidium bromide
staining dishes and shaker platform
camera for gel photography
ice water bath
BioRad smartspec and quartz cuvette
Methods:
RNA extraction.
1. Wash the monolayer cultures 2X with PBS just prior to extraction. Use 4-6 x
100mm dishes that have a confluent or slightly subconfluent monolayer of cells.
2. Remove all PBS from the dishes. Invert dishes and shake once or twice to remove
the last traces of PBS.
3. Add 8 ml of Trizol Reagent to the first dish. Allow to work for 10 sec, then
remove the cells with the scraper. After scraping cells in the first dish, pour the
contents into the second dish of cells. Allow to stand for 10 sec before scraping as
before. Repeat this procedure for all dishes. The cell lysate should become thick
and viscous after 2-3 dishes have been processed due to release of DNA.
4. Pour or pipette the cell lysate into a 15 ml polypropylene centrifuge tube. If the
lysate is viscous, pass the sample through a 25 gauge needle several times to shear
the high molecular weight DNA and reduce viscosity. The lysate should not stick
to a yellow tip when it is inserted into the solution. Make sure that there are no
clumps of indigested tissue.
5. Pour the 8 ml of cell lysate into a sterile round-bottom polypropylene centrifuge
tube. Add 1.6 ml of chloroform to the tube and replace the cap firmly. Shake the
tube vigorously for 30 sec to mix well.
6. Centrifuge the tube at 12,000 xg for 10 min at 2-8ºC. The sample should separate
into 2 distinct phases. The lower organic phase contains DNA and protein and the
upper aqueous phase has the RNA. Carefully remove the aqueous phase using a
sterile glass pipette. Do not take any of the organic phase or the interphase. Add
the aqueous phase to a clean sterile round-bottom polypropylene centrifuge tube.
7. Add 4.0 ml of isopropanol to the sample to precipitate the RNA. Incubate at room
temperature for 10 min. You should notice a cloudy white precipitate. Centrifuge
at 12,000 xg for 10 min at 2-8ºC.
8. After centrifugation, you should see a small white or amber colored pellet on the
bottom side of the centrifuge tube. Carefully pour the supernatant from the tube
and invert the tube on a paper towel to drain. Be careful that the RNA pellet does
not start to slide down the side of the tube.
9. Add 100-400 l of sterile deionized water to the tube and resuspend the RNA by
pipetting through a yellow or blue tip. Observe the solution carefully to see if all
of the RNA has been dissolved. This might require several min.
Measuring the RNA concentration in the sample:
1. Turn on the BioRad Smartspec (?) and set the analysis mode to DNA/RNA. Keep
all of your RNA samples on ice during this procedure. Push the button that says
RNA.
2. Set the blank for the spectrophotometer. Add 800 l of deionized water to a
quartz cuvette and place in the spec. Push the blank button.
3. Remove 10 l of RNA suspension and add to an eppendorf tube containing 1000
l of deionized water. Mix well by shaking the tube.
4. Add the contents to the cuvette and measure the absorbance and record the value.
Remove the cuvette and decant the sample. Briefly rinse the cuvette with water
before analyzing the next sample.
5. When finished, wash the quartz cuvette with water and invert to dry on a paper
towel.
6. calculate the concentration of RNA. Multiply the absorbance reading by 4000 (a
100 fold dilution of sample x 40 conversion factor) to get the concentration in
g/ml. Multiply this value by the sample volume (0.1-0.4 ml) to obtain the total
amount of RNA.
Analysis of RNA by electrophoresis through agarose gels containing formaldehyde:
1. Cast a 1.4% agarose gel containing MOPS buffer and formaldehyde. Add 4.2
grams of agarose, 30 ml of 10X MOPS buffer, and 240 ml of deionized water.
Microwave until the agarose is dissolved. Add 30 ml of 37% formaldehyde
solution and quickly close the top. Caution, formaldehyde is hazardous, do not
breath the fumes or get on the skin.
2. Place the bottle of agarose in a 65ºC water bath and wait several min until it is
cool to the touch.
3. Clean a minigel box and gel form. Tape the form to hold the agarose and insert
the comb. Pour a minigel containing approximately 70 ml of agarose. Quickly
replace the cover of the gel box and allow to polymerize for 30 min before use.
4. Hook up the leads of the gel box to the power pack. Make sure that the system
works by looking for bubbles coming from the wire electrode.
5. Remove your eppendorf tubes containing the precipitated RNA from the freezer
and place in an ice bath. Place samples in an eppendorf microcentrifuge and spin
for 10 min at top speed.
6. Examine the tube after the centrifuge has stopped. There should be a white or
amber pellet on the bottom side of each tube. If you have a pellet, pour away the
supernatant and invert the tube to dry on a paper towel. Wait at least 5 min.
7. While you are waiting, prepare a second set of eppendorf tubes, each containing 1
ml of deionized water.
8. Resuspend the RNA pellets in 100 – 400 l of sterile deionized water (depending
on the size of the pellet). Make sure that the solution of RNA is homogeneous. If
you have difficulty dissolving the RNA, add more sterile water and mix again.
9. Measure the absorbance at 260 nm as described previously. If the absorbance is
greater than 1.0, dilute the sample 10-fold and measure the absorbance again.
10. Calculate the volume of RNA solution to add for every well. Each well should
receive 10-20 g of RNA (10 g / RNA concentration in mg /ml = l / 10 g).
11. Set up an eppendorf tube for each lane of your gel. Place tubes on ice and add the
appropriate volume of RNA for 10-20 g / lane.
12. As soon as the RNA from all of the tubes has been sampled, add 1 ml of ethanol
and 30 ml of sodium acetate to the dissolved RNA. A precipitate should form
immediately. Return the RNA sample to the -70ºC freezer for long term storage.
13. Add 10-15 l of sample suspension buffer to each of the tubes containing the
RNA and mix well.
14. Place the tubes in a 65ºC water bath for 10 min to denature the RNA. Immediately
cool the tubes in an ice water bath for 5 min.
15. Add 7 l of gel loading buffer to each RNA sample and mix thoroughly. Load
sample into wells of the minigel and turn on the voltage to 90V. Allow the RNA
to run into the gel before you turn the voltage down to 60V. Run the minigel for
1-2 hours.
16. Remove the gel and rinse briefly in tap water. Place the gel in a dish with 0.5
mg/ml ethidium bromide and stain for 15 min. After staining, rinse the gel in tap
water and destain in deionized water fro 15 min. Photograph the gel for your
records. You should see 2 major bands in each lane (28S and 18S0 and the bands
should be sharp with no smearing of RNA. Smearing or tails of RNA indicate
RNA degradation.