Standard Operating Procedure
Title: Reverse Transcription of RNA for Quantitative Real-Time PCR
Department: Agronomy
Created by: Jason W. Haegele
Laboratory: Crop Production & Physiology Lab Suite
Supervisor: Dr. Mark Westgate
Lab Supervisor: Maria Hartt
Date approved: 13 March 2008
Procedure Overview: Gene expression analysis by quantitative real-time PCR requires two
steps. The first which is described in this standard operating procedure is reverse
transcription of RNA to cDNA. The resulting cDNA is amplified using a real-time PCR
instrument. See standard operating procedure “Gene Expression Analysis by Quantitative
Real-Time PCR” for instructions on the real-time PCR methods.
Equipment and reagents necessary:
Reagents:
RNA extracted and stored at -80oC
SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen: 18080-051), 1 kit
per 48 samples.
Kit contains:
Oligo(dT)20 primer (50 M), not used in this procedure
Random hexamer primers (50 ng/l)
10X RT buffer (200 mM Tris-HCL (pH 8.4), 500 mM KCl)
25 mM MgCl2
0.1 M dithiothreitol
10 mM dNTP mix
SuperScript III reverse transcriptase (200 U/ml)
RNaseOUT (40 U/ml)
E. coli RNase H (2 U/ml)
Diethyl pyrocarbonate treated water
Total HeLa RNA (10 ng/l)
Sense control primer (10 M)
Antisense control primer (10 M)
Platinum Taq DNA polymerase kit (Invitrogen:10966-018), 1 kit
Kit contains:
Platinum Taq DNA polymerase
10X PCR buffer minus Mg2+
50 mM MgCl2
50X TAE buffer (Eppendorf: 955155335), 10 ml per gel
Deionized water
Agarose, Broad Separation Range (Fisher: BP1356-100), 1.8 g per gel
Ethidium bromide (Fisher: AC17096-0010), 100 mg
10X DNA gel loading buffer (Eppendorf: 955155505), 5 l per reaction
Low DNA Mass Ladder (Invitrogen: 10068-013), 10 l per gel
Glassware:
10 ml graduated cylinder (1)
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100 ml graduated cylinder (1)
500 ml volumetric flask (1)
500 ml glass bottles (2)
Miscellaneous:
Eppendorf 5415D Microcentrifuge (1514 Agronomy)
Micropipettes and RNase free pipette tips: 10 l (Fisher: 05-403-72)
Thin-walled PCR tubes, 200 l (Fisher: E0030 124 260), 1 per sample
PCR tube racks
MJ Scientific DNA Engine (thermocycler, 1514 Agronomy)
Ice bucket and ice
-20oC freezer
Balance
Spatula
Weighing paper
15 ml conical tube (1)
Aluminum foil
Microwave
Waterbath
Bio-Rad Wide Mini-Sub electrophoresis unit and necessary components
Tape suitable for sealing ends of electrophoresis gel tray
Electrophoresis power unit
UV gel documentation system (1417 Agronomy Hall)
Procedure:
1. Gently mix the components of the SuperScript reverse transcription kit by inversion.
Briefly centrifuge the kit components in the Eppendorf microcentrifuge. This step and all
subsequent steps that require brief centrifugation are accomplished by holding the
“short” button on the Eppendorf microcentrifuge for approximately 10 seconds. This
generates enough centrifugal force to collect the tube contents in the bottom of the tube.
2. Use a pipette to combine the following components in a 0.2 ml PCR tube (1 tube per
sample):
Component
Amount
Sample RNA (volume should contain 5 g of total RNA)
n l
Primer: 50 ng/l random hexamers
1 l
10 mM dNTP mix
1 l
to 10 l
diethyl pyrocarbonate treated water
3. Prepare the positive and negative control reactions. Use a pipette to combine the
following components in 0.2 ml PCR tubes:
Component
Amount (+ RT control)
Amount (- RT control)
Diluted total HeLa RNA (100 pg/l)
1 l
1 l
Oligo(dT)20 primer
1 l
1 l
10 mM dNTP mix
1 l
1 l
diethyl pyrocarbonate treated water
7 l
7 l
4. Briefly centrifuge all PCR tubes in the Eppendorf microcentrifuge.
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5. Place the tubes in the thermocycler and incubate at 65oC for 5 minutes. Prior to using
the thermocycler, the reader of this SOP must review the instrument’s manual for
instructions on operation and programming.
6. Remove the tubes and place on ice for at least 1 minute.
7. Use a pipette to prepare the cDNA synthesis mix (sufficient for 10 sample reactions) by
combining the following components in a new 0.2 ml PCR tube:
Component
Amount
10X reverse transcriptase buffer
20 l
25 mM MgCl2
40 l
0.1M dithiothreitol
20 l
RNaseOUT
10 l
SuperScript III reverse transcriptase
10 l
8. Use a pipette to add 10 l of cDNA synthesis mix to each PCR tube containing the
sample RNA template/primer mix.
9. Mix the sample reaction tubes gently by inversion and centrifuge briefly to collect the
contents.
10. Place the tubes in the thermocycler and incubate for 10 minutes at 25oC.
11. Complete the control reactions while the sample reactions are incubating. Use a pipette
to add the following components to the tubes created in step 3:
Component
Amount (+ RT control)
Amount (- RT control)
10X reverse transcriptase buffer
2 l
2 l
25 mM MgCl2
4 l
4 l
0.1 M dithiothreitol
2 l
2 l
RNaseOUT
1 l
1 l
SuperScript III reverse transcriptase
1 l
-
diethyl pyrocarbonate treated water
-
1 l
12. Mix the control reactions gently by inversion. Centrifuge briefly to collect the contents.
13. Place the control reaction tubes in the thermocycler along with the sample reaction
tubes. Incubate for 50 minutes at 50oC.
14. Use the thermocycler to incubate the tubes for 5 minutes at 85oC to terminate the
reverse transcription reactions.
15. Remove the tubes and place on ice to chill.
16. Collect the tube contents by brief centrifugation.
17. Use a pipette to add 1 l of RNase H to each tube.
18. Collect the tube contents by brief centrifugation. Place in the thermocycler and incubate
at 37oC for 20 minutes.
19. Remove the tubes from the thermocycler. Store the tubes at -20oC. The cDNA from the
sample reactions will be quantified by real-time PCR using gene specific primers. If
ready, place the control reaction tubes on ice and continue to the next section of this
SOP.
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PCR of Control Reaction Products and Gel Electrophoresis
1. If they have been frozen, remove the control reactions from the -20oC freezer and place
on ice.
2. Use a pipette to add the following components to new 0.2 ml PCR tubes (one tube per
control reaction) sitting on ice:
Component
Amount
38.1 l
diethyl pyrocarbonate treated water
10X PCR buffer minus Mg
5 l
2+
1.5 l
50 mM MgCl2
10 mM dNTP mix
1 l
Control sense primer (10 M)
1 l
Control antisense primer (10 M)
1 l
cDNA from control RNA
2 l
0.4 l
Taq DNA polymerase (5 units/l)
3. Mix each tube gently by inversion. Centrifuge briefly to collect the contents.
4. Place the tubes in the thermocycler which has been preheated to 94oC. Incubate for 2
minutes at 94oC.
5. Perform 40 cycles of PCR with the following steps:

Denature
94oC for 15 seconds

Anneal
55oC for 30 seconds

Extend
68-72oC for 1 minute
6. Remove the tubes from the thermocycler and maintain at 4oC until ready to load agarose
gel.
Agarose Gel Electrophoresis of Control Reaction PCR Products:
1. 1X solution TAE Buffer: Using a graduated cylinder, add 10 ml 50X TAE reagent to a
properly labelled 500 ml volumetric flask; add enough deionized water to bring the final
volume to 500 ml. Use a funnel to transfer the buffer solution to an appropriately
labelled 500 ml glass bottle.
2. Ethidium bromide solution, 10 mg/ml: While wearing protective gloves and working in a
fume hood, weigh 100 mg of ethidium bromide. Place in a properly labelled 15 ml
conical tube wrapped in aluminium foil. Add 10 ml deionized water; thoroughly dissolve
the ethidium bromide. Retain excess solution at room temperature, avoiding exposure
to light and heat, for future use.
3. To make a 3% (w/v) agarose gel, weigh 1.8 g agarose powder; place in a properly
labelled 500 ml bottle. Using a graduated cylinder, measure 60 ml 1X TAE buffer; add to
bottle containing agarose powder. Completely dissolve agarose by placing bottle in
microwave oven for about 3-4 minutes on high power. Caution: agarose solution may
boil violently if overheated. While heating solution (and wearing an oven mitt),
periodically stop the microwave and gently swirl the bottle to ensure all agarose is being
dissolved. Once completely dissolved, carefully remove bottle from microwave and
allow to cool in a 55oC waterbath. (Caution: gel solution must be < 60oC at the time of
pouring to avoid damage to gel tray)
4. While gel is cooling, place tape over the ends of the Bio-Rad Wide Mini-Sub UVTP gel
tray. Level the gel tray on a horizontal surface.
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5. Position the comb on the walls of the UVTP gel tray. Adjust the comb with the aid of the
thumbscrews so that it remains 1 mm above the base of the UVTP tray.
6. Once the gel solution reaches 55oC, remove the bottle from the water bath and place in
the fume hood. Use a pipette to add 3 l of 10 mg/ml ethidium bromide solution to
bottle; mix thoroughly by gently swirling.
7. Avoiding the creation or air bubbles, slowly pour approximately 56 ml of liquid agarose
gel into the UVTP gel tray (gel should be ~ 4 mm thick). Allow the gel to solidify at room
temperature for up to 1 hour.
8. After the gel has solidified, carefully remove the tape and gel comb.
9. Place the UVTP gel tray into the Bio-Rad Wide Mini-Sub unit. Pour enough TAE buffer
into the unit to cover the gel by approximately 4 mm.
10. Using a pipette, add 5 l of 10X DNA gel loading buffer to each tube of control reaction
PCR product. Mix gently.
11. Using small/gel-loading tips, carefully pipette 10 l of each PCR product into designated
wells. Pipette 10 l of DNA size ladder into an adjacent well.
12. Place the lid on the electrophoresis system chamber – connecting the power cords to
the electrodes. Plug the opposite end of the leads into the power supply. Turn on the
power supply; set voltage to 60 V.
13. Run the gel until the bands are separated – about 1-2 hours.
14. Turn off the power supply, unplug leads and slide off the lid. While wearing gloves,
carefully remove the gel plate from the electrophoresis unit.
15. Record the PCR results using an ultraviolet light documentation system in 1417
Agronomy Hall. Note that the size of the PCR product from the positive control reaction
should be approximately 353-bp. There should be no visible band for the negative
control reaction.
16. Once finished, place the gel into a biohazard bag for autoclaving and the used buffer into
an appropriately labelled hazardous waste container. Place waste container(s) in the
Satellite Accumulation Area for disposal by ISU EH&S.
17. Wash all labware and clean all work areas. Rinse the electrophoresis unit components
under warm water. Allow to dry before reassembling for storage.
Personal Protective Equipment / Engineering Controls:
Nitrile gloves
Safety glasses
Face shield
Dust mask
Latex gloves
Splash goggles
Lab coat
Fume hood
Neoprene gloves
Vented goggles
Apron
Biosafety cabinet
Insulated gloves
Eye wash station
Safety shower
Respirator
Note: Open-toed and heeled shoes are NOT allowed.
Other Control Measures:
Handling & Storage Precautions:
Agarose: Store in a tightly closed container. Keep from contact with oxidizing materials. Keep
away from acids.
10X DNA Gel Loading Buffer: Store at 4oC in an appropriately labelled container.
Ethidium bromide: Keep container closed when not in use. Store in a cool, dry, well-ventilated
area away from incompatible substances.
Low DNA Mass Ladder: Store at -20oC in an appropriately labelled container.
Platinum Taq DNA Polymerase Kit Components: Keep in properly labelled containers. Store
at -20oC.
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SuperScript III Reverse Transcriptase Kit Components: Keep in properly labelled containers.
Store at -20oC.
TAE Buffer: Store at room temperature in an appropriately labelled container.
Waste Disposal 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.
Spill/Release Containment and Clean Up/Decontamination Procedures:
Agarose: Vacuum or sweep up material and place into a suitable disposal container.
10X DNA Gel Loading Buffer: Soak up with inert absorbent material.
Ethidium bromide: Vacuum or sweep up dry material and place into a suitable disposal
container. Absorb liquid spills with an inert material.
Low DNA Mass Ladder: Soak up with inert absorbent material.
Platinum Taq DNA Polymerase Kit Components: Soak up with inert absorbent material.
SuperScript III Reverse Transcriptase Kit Components: Soak up with inert absorbent material.
TAE buffer: Soak up with inert absorbent material.
Health & Safety Summary for Required Reagents:
The MSDS provided for many of Invitrogen’s reagents state that minimal risk is associated
with the concentration of chemical being used. As such, the health and safety summary
below is limited in scope for certain chemicals.
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Dithiothreitol
10X DNA Gel Loading
Buffer – Bromophenol
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“ “ – Xylene
Cyanol FF
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“ “ – EDTA, pH
8.0
“ “ – Glycerol
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Incompatibilities
Eyes.
Strong oxidizers, acids.
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N/A
N/A
0
0
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Eyes, skin,
respiratory tract,
gastrointestinal
tract.
Eyes, skin,
respiratory tract,
gastrointestinal
tract.
N/A
Strong oxidizing agents.
-
-
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Strong oxidizing agents.
-
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Strong oxidizing agents.
-
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Strong oxidizing agents.
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Eyes, skin,
respiratory tract,
gastrointestinal
tract.
N/A
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respiratory
tract
system.
dNTP mix
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Target Organ(s)
Agarose
Ethidium bromide
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HeLA RNA
X
Low DNA Mass Ladder
Eyes and skin.
Strong oxidizing agents.
-
-
-
N/A
N/A
0
0
0
Magnesium chloride
X
Eyes and skin.
N/A
0
0
0
10X PCR buffer
X
Eyes and skin.
1
0
0
Platinum Taq DNA
polymerase
Random hexamer
primers
RNase H
X
RNaseOUT
10X RT buffer
N/A
N/A
1
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N/A
N/A
0
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0
N/A
N/A
-
-
-
N/A
N/A
1
1
0
N/A
N/A
-
-
-
Sense/Antisense
control primers
SuperScript III Reverse
Transcriptase
X
Eyes and skin.
Strong oxidizing agents.
-
-
-
X
Strong oxidizing agents.
-
-
-
TAE buffer
X
Eyes, skin,
respiratory tract,
kidneys.
Eyes, skin, and
respiratory tract.
Strong oxidizing agents.
-
-
-
 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:
Sambrook, J., and D. Russell. 2001. Molecular Cloning: A Laboratory Manual, Volume 1
(third Edition). CSHL Press.
Product manuals for SuperScript III Reverse Transcription Kit and Platinum Taq DNA
Polymerase Kit.
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