Standard Operating Procedure
Title: Extraction of RNA and DNA from maize endosperm and embryo tissue
Department: Agronomy
Created by: Jason Haegele
Laboratory: Crop Production & Physiology
Supervisor: Dr. Mark Westgate
Lab Supervisor: Maria Hartt
Date approved: 25 February 2008
Procedure Overview:
The primary goal of this procedure is to extract total RNA from maize
endosperm for gene expression profiling by quantitative real-time reverse-transcription PCR.
Starch and protein are separated from nucleic acids using sodium dodecyl sulfate (SDS). RNA is
separated and precipitated from the remaining mixture using the TRIZOL reagent from Invitrogen.
DNA is also recovered using TRIZOL and can be used for normalizing RNA yields from sample to
sample.
Equipment and reagents necessary:
Aluminum foil
RNaseZAP (Sigma: R2020-250ML)
Diethyl pyrocarbonate (Fisher: AC17025-0050)
Micropipettors
RNase-free pipette tips: 10 l (Fisher: 05-403-72), 200 l (Ambion: 12650), and 1000 l (Ambion:
12660)
Deionized water
1000 ml volumetric flask and glass bottles for preparing DEPC treated water
Funnels
Autoclave
Tris(hydroxymethyl) aminomethane (BioRad: 161-0716)
Balance
Weighing paper
Spatulas
500 ml volumetric flasks (3)
500 ml glass beaker (1)
Magnetic stir bars and stir plate
pH meter and calibration buffer solutions
32-38% hydrochloric acid
500 ml glass bottles (4)
Lithium chloride
Sodium dodecyl sulfate (Acros: 41953-1000)
Ethylenediaminetetraacetic acid, 0.5M, pH 8.0 (Ambion: 9260G)
100 ml volumetric flask (1)
100 ml glass bottle (1)
Sodium citrate dihydrate (Fisher: S279-500), 0.1M, in 10% (v/v) ethanol, 2 ml per sample
250 ml glass beaker (1)
500 ml graduated cylinder (1)
Ethanol, 100% (v/v), 300 l per sample
Sodium chloride, 5M, (Fisher: S271-500), 30.6 l per sample
50 ml volumetric flask (1)
50 ml glass bottle (1)
1
24 February 2008 jwh
Frozen (-80oC) endosperm or embryo tissue
Liquid nitrogen
Receptacle for liquid nitrogen
Forceps, large and small
1.5 ml RNase-free microcentrifuge tubes (Ambion: 12400), 3 per sample
RNase-free plastic pestle (Fisher: K749515-1500)
Phenol:chloroform:isoamyl alcohol, pH 7.9±0.2 (Ambion: 9730), 400 l per sample
Vortex
Ice bucket and ice
Phase Lock Gel Tube, 2 ml, heavy formulation (Fisher: FP2302830), 1 per sample
Sorvall RT6000 Refrigerated Centrifuge (1514A Agronomy)
Chloroform (Fisher: C298SK-4), 400 l per sample
Microcentrifuge tube racks
TRIZOL reagent (Invitrogen: 15596-026), 1 ml per sample
Ammonium acetate, 5M, (Ambion: AM9070G), 50 l per sample
Glycoblue coprecipitant, (Ambion: AM9515), 1.7 l per sample
Isopropyl alcohol (Fisher: S77795), 500 l per sample
RNA Storage Solution (Ambion: 7001), 50 l per sample
-80 freezer
Ethanol, 75% (v/v), 2.5 ml per sample
Tris-EDTA (TE) buffer, pH 8.0 (Ambion: AM9849), 1 ml per sample
RNase Cocktail Enzyme Mix (Ambion: AM2286), 1 l per sample
Eppendorf 5415D Microcentrifuge (1514 Agronomy)
Tongs
Heating block
Ethanol, 95% (v/v), 1 ml per sample
Steps for Avoiding RNase Contamination:
1. Wear gloves at all times when working with samples.
2. Glass items should be baked at 150oC for 4 hours in the ovens in 1426 Agronomy Hall. Use
aluminum foil to cover the tops of bottles, beakers, flasks, etc. to prevent contamination after
removing from oven. Wear oven mitts and exercise caution as glass items will be hot.
3. Work surfaces, plastic items, forceps, and other non-disposable items must be treated with
RNaseZAP according to the manufacturer’s instructions.
Solution Preparation:
Work in a fume hood and wear appropriate protective apparel while preparing solutions.
1. Diethyl pyrocarbonate (DEPC) treated water (RNase-free): All solutions should be made with
DEPC treated water. DEPC is an inhibitor of RNase. Use a pipette to add 1000 l of DEPC to
approximately 500 ml of deionized water in a 1 L volumetric flask. Bring to volume with
additional deionized water. Use a funnel to transfer the solution to appropriately labeled 1 L
glass bottle. Allow to sit overnight and then autoclave at 121oC for 1 hour to inactivate the
DEPC.
2. Prepare a 0.5M solution of Tris(hydroxymethyl) aminomethane by using a scale to weigh 30.3 g
of Tris. Use a funnel to transfer the Tris to a 500 ml volumetric flask containing a small amount
of DEPC treated water. Swirl to dissolve. Bring to volume with additional DEPC treated water.
Transfer to a 500 ml beaker, insert a magnetic stir bar, and place on a stir plate. Use the pH
2
24 February 2008 jwh
meter and 32-38% hydrochloric acid (add dropwise) to adjust the pH to 8.0. Use a funnel to
transfer the solution to a properly labeled 500 ml glass bottle.
3. Prepare a 1M solution of lithium chloride by using a scale to weigh 21.2 g of lithium chloride.
Use a funnel to transfer the lithium chloride to a 500 ml volumetric flask containing a small
amount of DEPC treated water. Swirl to dissolve. Bring to volume with additional DEPC treated
water. Use a funnel to transfer the solution to a properly labeled 500 ml glass bottle.
4. Prepare a 5% (w/v) solution of sodium dodecyl sulfate (SDS) by using a scale to weigh 25 g of
SDS. Use a funnel to transfer the SDS to a 500 ml volumetric flask containing a small amount
of DEPC treated water. Swirl to dissolve. Bring to volume with additional DEPC treated water.
Use a funnel to transfer the solution to a properly labeled 500 ml glass bottle.
5. Prepare the RNA/DNA extraction buffer by using a pipette to transfer 10 ml of 0.5 M Tris, 15 ml
of 1M lithium chloride, 1 ml of 0.5M EDTA, and 20 ml of 5% (w/v) SDS to a properly labeled 100
ml volumetric flask. Bring to volume with additional DEPC treated water. Use a funnel to
transfer the solution to a properly labeled 100 ml glass bottle. The final concentrations in this
solution are: 50mM Tris, 150mM lithium chloride, 5mM EDTA, and 1% (w/v) SDS.
6. Prepare a 0.1M solution of sodium citrate containing 10% (v/v) ethanol by using a scale to
weigh 16.8 g of sodium citrate dihydrate. Transfer the sodium citrate dihydrate to a 250 ml
glass beaker containing approximately 200 ml of deionized water. Dissolve using a magnetic
stir bar and stir plate. Use a funnel to transfer the sodium citrate solution to a 500 ml graduated
cylinder. Use a funnel to add 50 ml of 100% ethanol. Bring to volume with additional deionized
water. Use a funnel to transfer to a properly labeled 500 ml glass bottle.
7. Prepare a 5M solution of sodium chloride by using a scale to weigh 14.6 grams of sodium
chloride. Use a funnel to transfer the sodium chloride to a 50 ml volumetric flask containing a
small amount of deionized water. Swirl to dissolve. Bring to volume with additional deionized
water. Use a funnel to transfer to the solution to a properly labeled 50 ml glass bottle.
Protocol:
Sodium dodecyl sulfate extraction:
1. All steps involving phenol, chloroform, or TRIZOL reagent should be performed while
working in the fume hood. Special care should be taken when transferring or using
liquid nitrogen including the use of insulated gloves. Liquid nitrogen should not be
poured into the black laboratory sinks as this may crack them.
2. Remove the endosperm/embryo samples needed for RNA/DNA extraction from the -80oC
freezer. Place the sample tubes in a bucket of liquid nitrogen. Using large forceps remove
each tube (one at a time) from the liquid nitrogen and open. Using small forceps transfer
approximately 100 mg of endosperm or embryo tissue to a labeled RNase-free microcentrifuge
tube. Place new tubes containing 100 mg of tissue in the liquid nitrogen. Return the original
tissue samples to the -80 freezer.
3. Steps 4 and 5 should be performed for one sample at a time. Once a manageable
number of samples (~8) are complete, proceed to step 6 and continue simultaneously
with all ground samples.
4. Remove each tube from the liquid nitrogen and use a pipette to add 200 l of RNA/DNA
extraction buffer.
5. Quickly grind the tissue using a RNase-free plastic pestle.
6. Use a pipette to add 200 l of phenol:chloroform:isoamyl alcohol to each tube. Cap each tube
and vortex briefly. Place the tubes on ice for approximately 5 minutes. Mix occasionally by
inverting the tubes.
7. Use a pipette to transfer the solution from each tube to labeled Phase Lock Gel tubes.
3
24 February 2008 jwh
8. Centrifuge the tubes in the Sorvall centrifuge for 20 minutes at 3,400 rpm and 4oC.
9. Use a pipette to add an additional 200 l of phenol:chloroform:isoamyl alcohol. Cap each tube
and vortex briefly.
10. Centrifuge the tubes for 20 minutes at 3,400 rpm and 4oC.
11. Use a pipette to add 200 l of chloroform to each tube. Cap each tube and vortex briefly. Place
the tubes on ice for 5 minutes. Mix occasionally by inverting the tubes.
12. Centrifuge the tubes for 20 minutes at 3,400 rpm and 4oC.
TRIZOL separation of nucleic acids:
13. Pour the aqueous phase from each PHASE LOCK tube (it will be on top) into labeled RNasefree microcentrifuge tubes. Place tubes in rack.
14. Use a pipette to add 1 ml of TRIZOL reagent to each microcentrifuge tube; cap.
15. Shake each tube well for 15 seconds and then incubate for 5 minutes at room temperature.
16. Use a pipette to add 200 l of chloroform to each tube. Cap tubes and shake vigorously by
hand for 15 seconds. Incubate for 2-3 minutes at room temperature.
17. Centrifuge the tubes for 45 minutes at 3,400 rpm and 4oC.
Precipitation and preparation of RNA:
18. Use a pipette to transfer the upper aqueous phase to a new, labeled RNase-free
microcentrifuge tube. Place tubes with aqueous phase in a separate rack. Retain the tubes
containing the lower organic phase for DNA precipitation at 4oC for processing the next day.
19. Use a pipette to add 50 l of 5M ammonium acetate to each tube containing the aqueous
phase.
20. Use a pipette to add 1.7 l of glycoblue to each tube.
21. Use a pipette to add 500 l of isopropyl alcohol to each tube containing the aqueous phase.
Cap and shake well. Incubate for 15 minutes at -20oC.
22. Centrifuge the tubes for 30 minutes at 3,400 rpm and 4oC. RNA precipitate should be visible.
23. Gently pour the supernatant into waste container without disturbing the pellet. Use a pipette to
add 1 ml of 75% ethanol (made with DEPC treated water) to each tube.
24. Centrifuge the tubes for 10 minutes at 3,400 rpm and 4oC.
25. Gently pour the supernatant into waste container without disturbing the pellet.
26. Air dry the RNA pellet for approximately 5-10 minutes.
27. Use a pipette to add 50 l of RNA storage solution (Ambion). Vortex until pellet is completely
dissolved. Place tubes in a labeled, closed container and store at -80oC. RNA will be used for
gene expression profiling by reverse-transcription real-time PCR.
Precipitation and preparation of DNA:
28. Use a pipette to add 300 l of 100% ethanol to each of the tubes containing the organic phase
from step 18. Cap the tubes and mix by inversion. Incubate the tubes at room temperature for
2-3 minutes.
29. Centrifuge the tubes for 5 minutes at 3,000 rpm and 4oC. Gently pour the supernatant into a
waste container.
30. Use a pipette to add 1 ml of 0.1M sodium citrate in 10% ethanol. Incubate at room temperature
for 30 minutes. Mix periodically by inversion.
4
24 February 2008 jwh
31. Centrifuge the tubes for 5 minutes at 3,000 rpm and 4oC. Gently pour the supernatant into
waste container.
32. Repeat steps 30-31 once more.
33. Use a pipette to add 1.5 ml of 75% ethanol. Incubate at room temperature for 10-20 minutes.
Mix periodically by inversion.
34. Centrifuge the tubes for 5 minutes at 3,000 rpm and 4oC. Gently pour the supernatant into
waste container.
35. Air dry the DNA pellet for 5 to 15 minutes.
36. Using a pipette, add 500 l of TE buffer (pH 8.0); cap tube. Vortex to completely dissolve the
DNA pellet.
37. Using a pipette, place a 1 l drop of RNase Cocktail Enzyme Mix on the side of the tube; cap
tube.
38. Place the tubes in the Eppendorf microcentrifuge; spin briefly to mix the contents of the tube.
39. Using tongs, place the tubes in the heating block and incubate at 37oC for 30 minutes.
40. Using tongs, remove the tubes from the heating block and place in rack. Using a pipette, add
30.6 l of 5M sodium chloride and 1 ml of 95% tax free ethanol; cap tube.
41. Mix well by inversion and incubate at room temperature for 10 minutes.
42. Centrifuge the tubes for 5 minutes at 3,000 rpm and 4oC. Gently pour the supernatant into a
waste container.
43. Air dry the DNA pellet for 5 to 15 minutes.
44. Using a pipette, add 500 l of TE buffer (pH 8.0). Vortex to completely dissolve the DNA pellet.
45. Place tubes in a labeled, closed container and store at -20oC. DNA will be used for testing
primers and normalizing RNA extraction yields.
Personal Protective Equipment / Engineering Controls:
Eye protection (goggles & shield)
Skin protection (proper shoes, gloves, lab coat, etc.)
Ventilation system
Safety shower
Eye wash station
Hazard Controls & Storage Precautions:
Ammonium acetate: Keep in a tightly closed container in a cool, dry, ventilated area. Protect against
physical damage. Isolate from incompatible substances.
Chloroform: Do not store in direct sunlight. Store in a cool, dry, well-ventilated area away from
incompatible substances. Keep away from acids, alkaline substances and strong mineral acids.
Diethyl pyrocarbonate: Keep away from sources of ignition. Store in a tightly closed container in a
dry area. Keep refrigerated. (Store at 4°C)
Ethanol: Avoid breathing vapor, contact with eyes, skin, and clothing. Avoid prolonged or repeated
exposure. Store away from heat, sparks, and open flame in a tight container in a cool, dry
place.
Ethylenediaminetetraacetic acid (EDTA): Keep in a tightly closed container, stored in a cool, dry,
ventilated area. Protect against physical damage. Isolate from incompatible substances.
Glycoblue: Isolate from incompatible substances.
Hydrochloric acid: Use under fume hood. Use only with adequate ventilation or respiratory
protection. Do not use with metal spatula or other metal items. Wash thoroughly after handling.
5
24 February 2008 jwh
Remove contaminated clothing and wash before reuse. Use caution when opening. Contents
may develop pressure upon prolonged storage. Do not get in eyes, on skin, or on clothing. Do
not breathe vapor or mist, ingest or inhale. Discard contaminated shoes. Keep container tightly
closed and away from strong bases, metals, alkalis and oxidizing materials. Store in a cool, dry,
well-ventilated corrosives area away from incompatible substances. Do not store in metal
containers.
Isopropyl alcohol: Peroxidizable substance. Keep away from heat, sparks, and flame. Do not
store in direct sunlight. Store in a tightly closed container in a cool, dry, well-ventilated area
away from incompatible substances and protected from moisture and ignition sources.
Flammables-area. Keep from contact with oxidizing materials. After opening, purge container
with nitrogen before reclosing. Periodically test for peroxide formation on long-term storage.
Addition of water or appropriate reducing materials will lessen peroxide formation. Containers
should be dated when opened and tested periodically for the presence of peroxides. Should
crystals form in a peroxidizable liquid, peroxidation may have occurred and the product should
be considered extremely dangerous. In this instance, the container should only be opened
remotely by professionals.
Liquid nitrogen: Keep in a well-ventilated room.
Lithium chloride: Do not store in direct sunlight. Store in a tightly closed container in a cool, dry,
well-ventilated area away from incompatible substances and protected from moisture.
Phenol:Chloroform:Isoamyl alcohol: Keep away from heat, sparks, flames, sources of ignition and
moisture. Store at 4oC in amber or foil-wrapped bottles. To avoid oxidation and breakdown
products, do not open frequently. Keep from contact with oxidizing materials.
RNA Storage Solution: Store at 4oC.
RNase Cocktail Enzyme Mix: Store at -20oC.
RNaseZAP: Store at room temperature.
Sodium citrate dihydrate: Wash thoroughly after handling. Remove contaminated clothing and
wash before reuse. Use with adequate ventilation. Minimize dust generation and accumulation.
Avoid contact with eyes, skin, and clothing, ingestion and inhalation. Keep from contact with
oxidizing materials. Store in a tightly closed container in a cool, dry, well-ventilated area away
from incompatible substances.
Sodium chloride: Store in a cool, dry place in a tightly closed container.
Sodium dodecyl sulfate: Keep away from sources of ignition. Store in a cool, dry place in a tightly
closed container. Flammables-area.
Tris-EDTA buffer: Store at room temperature.
Tris(hydroxymethyl) aminomethane: Store at room temperature in a tightly closed container in a
cool, dry, well-ventilated area away from incompatible substances. Keep away from acids. Store
protected from moisture.
TRIZOL reagent: Store at 4oC.
Waste Disposal & Decontamination Procedures:
Unless EH&S specifically instructs otherwise, all chemical/reagent waste (including excess
solutions) must be placed in an appropriately labeled hazardous waste container for EH&S
disposal. Compatible substances may be combined into one waste container.
6
24 February 2008 jwh
Health & Safety Info for Required Reagents
C
a
r
c
i
n
T
e
r
a
t
o
g
e
n
C
o
r
r
o
s
i
v
e
T
o
x
i
c
I
r
r
i
t
a
n
t
o
S
e
n
s
i
t
i
z
e
r
g
H
i
g
h
l
y
C
o
m
b
u
s
t
i
b
l
e
T
o
x
i
c
n
Ammonium acetate
Chloroform
X
X
X
X
X X
F
l
a
m
m
a
b
l
e
O
r
g
a
n
i
c
P
e
r
o
x
i
d
e
s
O
x
i
d
i
z
e
r
P
y
r
o
p
h
o
r
i
c
U
n
s
t
a
b
l
e
W
a
t
e
r
H
e
a
l
t
h
F
l
a
m
m
a
b
i
l
i
t
y
R
e
a
c
t
i
v
i
t
y
R
e
a
c
t
i
v
e
Target Organ(s)
Incompatibilities
Eyes, skin,
respiratory tract,
gastrointestinal
system.
Kidneys, heart,
central nervous
system, liver, eyes,
reproductive
system, skin.
Sodium hypochlorite and
strong acids.
1
1
0
Alkali metals, fluorine,
caustics (e.g. ammonia,
ammonium hydroxide,
calcium hydroxide,
potassium hydroxide,
sodium hydroxide),
dinitrogen tetraoxide,
sodium hydroxide +
methanol, potassium tertbutoxide, chemically
active metals, powdered
aluminum, nitrogen
tetroxide, powdered
magnesium, acetone +
alkali, disilane, perchloric
acid + phosphorus
pentoxide, sodium
methylate,
triisopropylphosphine.
Ammonia, strong acids,
strong bases, strong
oxidizing agents, strong
reducing agents.
Strong oxidizing agents,
acids, alkali metals,
ammonia, hydrazine,
peroxides, sodium, acid
anhydrides, calcium
hypochlorite, chromyl
chloride, nitrosyl
perchlorate, bromine
pentafluoride, perchloric
acid, silver nitrate,
mercuric nitrate,
potassium-tert-butoxide,
magnesium perchlorate,
acid chlorides, platinum,
uranium hexafluoride,
silver oxide, iodine
heptafluoride, acetyl
bromide, disulfuryl
difluoride,
tetrachlorosilane + water,
acetyl chloride,
permanganic acid,
ruthenium (VIII) oxide,
uranyl perchlorate,
potassium dioxide.
Oxidizing agents.
2
0
0
2
2
1
2
3
0
1
0
0
Oxidizers
1
1
0
Diethyl pyrocarbonate
X
Central nervous
system, respiratory
system, eyes, skin.
Ethanol
X X
Kidneys, heart,
central nervous
system, liver.
Ethylenediaminetetra
acetic acid
Glycoblue
X
Respiratory
system, eyes, skin.
Eyes, skin,
respiratory tract.
X
E
x
p
l
o
s
i
v
e
G
a
s
e
Chemical name
C
o
m
p
r
e
s
s
e
d
X
X
7
24 February 2008 jwh
Hydrochloric acid
X
Isopropyl alcohol
X
Respiratory
system,
gastrointestinal
system, teeth,
eyes, skin.
X
Central nervous
system, respiratory
system, eyes, skin.
Liquid nitrogen
Respiratory
system, exposed
tissues.
Lithium chloride
Phenol:chloroform:
isoamyl alcohol
X
X
X
X
RNA Storage Solution
(see sodium citrate)
RNase cocktail
enzyme mix
RNaseZAP
X
Sodium chloride
X
Sodium citrate
dihydrate
Sodium dodecyl
sulfate
Tris(hydroxymethyl)
aminomethane
X
Tris-EDTA buffer
TRIZOL reagent
X
X
X
X
X
Central nervous
system, eyes, skin.
X
Eyes, skins,
respiratory tract,
liver, kidneys,
central nervous
system.
X
Metals, strong oxidizing
agents, strong reducing
agents, bases, acetic
anhydride, alcohols,
amines, sulfuric acid,
vinyl acetate, epoxides
(e.g. butyl glycidyl ether),
chlorosulfonic acid,
carbides, betapropiolactone,
ethyleneimine, propylene
oxide, lithium silicides, 2aminoethanol, 1,1difluoroethylene,
magnesium boride,
mercuric sulfate,
aldehydes, cyanides,
sulfides, phosphides.
Strong oxidizing agents,
strong acids, strong
bases, amines, ammonia,
ethylene oxide,
isocyanates,
acetaldehyde, chlorine,
phosgene, Attacks some
forms of plastics, rubbers,
and coatings., aluminum
at high temperatures.
Reacts with oxygen and
hydrogen on sparking
forming nitric oxide and
ammonia. Combines
directly with lithium and at
red heat with calcium,
strontium and barium to
form nitrides. Forms
cyanides when heated
with carbon in presence
of alkalies or barium
oxides.
Strong oxidizing agents,
strong acids, alkali
metals, bromine
trifluoride, halogens,
sulfuric acid.
Each component has
specific incompatibilities;
review MSDS.
3
0
1
1
3
0
3
0
0
2
1
1
3
2
1
N/A
N/A
Eyes, respiratory
system, and skin.
Eyes, skin, and
respiratory tract.
Acids, Oxidizing agents,
Reducing agent, Bases.
Metals, strong oxidizing
agents, strong acids,
bromine trifluoride, nitro
compounds,
dichloromaleic anhydride
+ urea.
Oxidizing agents.
2
1
1
1
1
1
1
0
0
Respiratory system, eyes,
skin.
Strong oxidizing agents,
strong acids.
2
3
0
2
1
0
?
?
?
Eyes, skin, and
respiratory tract.
Respiratory
system, eyes, skin.
Eyes, skin, upper
respiratory tract,
kidneys, liver.
N/A
Eyes, skin, and
respiratory tract.
8
X
N/A
Strong oxidizing agents.
Strong acids.
24 February 2008 jwh
 The above summary consists of guidelines for proper handling & disposal of chemicals
used in this procedure. You must read and understand the contents of the entire
MSDS(s) before starting this procedure.
References:
Leiva, J., R. Dante, and D. Holding. 2002. RNA extraction from maize endosperm using SDSTRIZOL protocol. Larkins Lab Protocol (University of Arizona). Available online at:
http://www.ag.arizona.edu/research/larkinslab/protocols/RNA%20extraction%20from%20endos
perm%20-SDS-Trizol%20combo.pdf. Last accessed: 2 January 2008.
Invitrogen.
2007.
TRIZOL
reagent
manual.
Available
online
at:
http://www.invitrogen.com/content/sfs/manuals/15596018%20pps%20Trizol%20Reagent%2006
1207.pdf. Last accessed: 2 January 2008.
9
24 February 2008 jwh