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RNA amplification protocol
This method is to generate from minute amount of total RNA, sufficient amount of mRNA to
perform microarray experiments
You will need:
Total RNA
If possible use 3 µg,
For a better success, clean the Trizol
extracted total RNA using the RNeasy
kit.
To Save columns: If your plan is to
pool your RNA samples. Pool first
than purify
- Using less than 0.5 µg of starting
RNA is more challenging, you will
need to perform a second round of
amplification.
- 1 µg is enough
- More than 2 µg is theoretically better
Oligo-dT-T7
primer
5' - GCA TTA GCG GCC GCG AAA TTA
ATA CGA CTC ACT ATA GGG AGA TTT
TTT TTT TTT TTT TTT TTT V - 3'
Make a 500 ng/µl solution, use RNase free
water
Random
hexamers
Mmlv
- Must be PAGE Purification
- Reference: Baugh LR, Hill A. A.,
Brown E. L. and Hunter CP (2001)
Quantitative analysis of mRNA
amplification by in vitro transcription .
Nucleic Acids Research, Vol. 29, No.
5 e29.
5’ NNNNNN 3’
Make a 1µg/µl solution, use RNase free water
Promega 200 units/µl
Superscript II or
III
Invitrogen 200 units/µl
dNTPs
rNTPs
E.coli DNA
polymerase
E.coli Rnase H
E.coli DNA
ligase
T4 DNA
polymerase
T7 RNA
polymerase
Pyrophosphatase
HEPES 1M pH
7.5
ISC Bioexpress, make 10 mM stocks
NEB (80 mM solutions, use as it is)
NEB, 10 units/µl, works with NEB buffer #2
Takara (2150A), 60 units/µl
NEB, 10 units/µl
NEB, 3 units/µl
Fermentase, 200 units/µl
Fermentas, 0.1 units/µl
Good enough, cheaper than the
superscript II
Most used/cited in articles
Is supposed to generate full length
cDNAs
Spermidine
100mM
Dithiothreitol
(DTT) 1M
Acetyled BSA
RNeasy kit
PCR qiaquick
purification kit
RNase free
Water
Filter tips
Speed vacuum
PCR machine
Make 100 mM stocks, store at -20C
Make 1M stocks, store at -20C
10mg/ml (100X)
Qiagen
Qiagen
Use the RNase free water that comes with the
RNeasy kit
Is in the Genelab
Incubate your reaction in 0.2 ml tubes
Set the heated lid at 37C when
performing the overnight invitro
transcription
There are three steps:
1. Step1:
First strand cDNAs synthesis by reverse transcription (the products are single stranded cDNAs).
The T7 promoter is added to the cDNAs synthesized by the use of the oligo-dT-T7 primer.
2. Step2:
Second strand synthesis (the products are double stranded DNA molecules)
The T7 promoter need to be double stranded to be functional.
3. Step 3:
The Invitro transcription: the T7 RNA polymerase will transcribe the cDNAs into mRNAs .
If necessary (you do not obtain a sufficient yield for all the microarray hybridizations you plan),
perform a second round of amplification.
1. Step1: First strand cDNAs synthesis
Follow the next procedures, use 0.2 ml PCR tubes, filter tips, do the incubation in the PCR
machine .
Total RNA
Oligo-dT-T7 primer (500ng/µl)
H2O
3 µg
1 µl
Up to 15 µl
Incubate for 5 min at 70 C, then immediately chill on ice (leave on ice for 10 min).
Spin the tubes, and add 10 µl of the following mixture (prepare a master mix for the number of
samples you want to amplify): Final volume is 25 µl.
5 X First strand Buffer (contain
DTT)*
dNTPs 10 mM
MMLV (200 units/µl)
RNase inhibitor
H2O
5 µl
1.25 µl
1 µl
0.5 µl
2.25 µl
* some buffer do not contain DTT, in this case add DTT to 10 mM final concentration
Mix gently by pipetting up and down, and incubate at 42C for 1hour to 1hour 30min.
Inactivate the reverse transcriptase by incubating the reaction at 70C for 10 min.
Spin the tubes.
2. Step2: Second strand synthesis (the products are double stranded DNA molecules)
Add 75 µl of the following mixture to the RT reactions: Final volume is 100 µl
E.coli DNA polymerase buffer (10X) (use the
10 µl
NEB buffer #2)
dNTPs 10 mM
2.5 µl
E.coli Rnase H (60 units/µl)
0.05 µl (3 units)
E.coli DNA polymerase (10 units/µl)
4 µl (40 units)
H2O
Up to 75 µl
Incubate at 16C for 2hours
Then, add:
E.coli DNA ligase
DNA ligase buffer
10x
0.1µl (1 unit)
11 µl
Incubate at RT for 15 min
Then add:
T4 DNA polymerase
2 µl (6 units)
Incubate for 15 min at RT
Purify the double strand cDNAs using the qiagen qiaquick PCR purification kit.
To improve the elution yield, use a bigger volume of H2O (two times 50 µl) than mentioned in
the qiagen manual.
Speed-vacuum to dry.
3. Step 3: Invitro transcription
Add 40 µl of the following invitro transcription cocktail to the double strand cDNAs that you
speed vacuumed:
HEPES 1 M pH 7.5
4.8 µl (120 mM)
MgCl2 2M
0.56 µl (28 mM)
rNTPs 80 mM
3 µl (6 mM)
Spermidine 100 mM
0.4 µl (1 mM)
Acetyled BSA 10X
0.4 µl (0.1 mM)
DTT 1M
1.6 µl (40 mM)
RNase inhibitor
0.5 µl
Pyrophosphatase
2 µl (0.2 units)
T7 RNA polymerase
1 µl (200 units)
H2O
25.74 µl
Incubate for 14 hours at 37 C (SET THE PCR MACHINE HEATED LID TEMPERATURE
AT 37C)
Purify the amplified RNA using the qiagen RNeasy kit.
Elute two times with 50 µl RNase free water each time.
Speed vacuum to concentrate (for example until 30 µl are left)
Quantify using the nanodrop
If you did not generate a sufficient amount of amplified RNA, perform a second round of
amplification.
Second round of amplification
Carry out the 3 above steps, but with the following modifications:
Step1:
Perform the RT reactions using the random hexamers primers (5’NNNNNN3’) instead of the
oligo-dT-T7 primer
Total RNA
3 µg
Random primers 1µg/µl
1 µl
H2O
Up to 15 µl
Incubate at 70C for 5 min then immediately chill on ice and leave for 10 min at room
temperature.
Spin the tubes, and add 10 µl of the following mixture (prepare a master mix for the number of
samples you want to amplify):
5 X First strand Buffer (contain
DTT)*
dNTPs 10 mM
MMLV (200 units/µl)
RNase inhibitor
H2O
5 µl
1.25 µl
1 µl
0.5 µl
2.25 µl
* some buffer do not contain DTT, in this case add DTT to 10 mM final concentration
Mix gently by pipetting up and down, and incubate at 37 C for 1 hour.
Then, incubate for 2 min at 94 C, chill rapidly on ice.
Spin the tubes.
2. Step2: Second strand synthesis (the products are double stranded DNA molecules)
Add:
Oligo-dT-T7 primer (500 ng/µl)
1 µl
Incubate at 70 C for 5 min, then 3 min at 42 C
Add 74 µl of the following mixture (final volume = 100 µl)
E.coli DNA polymerase buffer (10X) (use the
10 µl
NEB buffer #2)
dNTPs 10 mM
2.5 µl
E.coli Rnase H
0.05 µl (3 units)
E.coli DNA polymerase
4 µl (40 units)
H2O
Up to 74 µl
Incubate at 16C for 2hours
Then, add:
T4 DNA polymerase
2 µl (6 units)
Incubate at RT for 15 min
Purify the double strand cDNAs using the qiagen qiaquick PCR purification kit.
Speed vacuum until dry
3. Step 3: Invitro transcription
Add 40 µl of the following invitro transcription cocktail to the double strand cDNAs that you
speed vacuumed:
HEPES 1 M pH 7.5
MgCl2 2M
rNTPs 80 mM
Spermidine 100 mM
Acetyled BSA 10X
DTT 1M
RNase inhibitor
Pyrophosphatase
T7 RNA polymerase
H2O
4.8 µl (120 mM)
0.56 µl (28 mM)
3 µl
0.4 µl (1 mM)
0.4 µl (0.1 mM)
1.6 µl (40 mM)
0.5 µl
2 µl (0.2 units)
1 µl (200 units)
25.74 µl
Incubate for 14 hours at 37 C (SET THE PCR MACHINE HEATED LID TEMPERATURE
AT 37C)
Purify the amplified RNA using the qiagen RNeasy kit.
Elute two times with 50 µl RNase free water each time
Speed vacuum to concentrate (for example until 30 µl are left)
Quantify using the nanodrop
Amplified RNA labeling for microarray
1. Reverse transcription reactions
Do not forget: Use the random hexamer primers instead of the oligo-dT primer
Amplified RNA
Random primers 1µg/µl
H2O
3.475 µg
1 µl
Up to 15 µl
Incubate for 10 min at 70 C, chill immediately on ice.
Add 10 µl of following mixture (final volume = 25 µl)
5 X First strand Buffer (contain
5 µl
DTT)*
Amino allyl dNTPs 10 mM
1.25 µl
(dATP 10 mM, dGTP 10 mM, dCTP 10 mM,
dTTP 3.33 mM, amino allyl dUTP 6.66 mM)
MMLV (200 units/µl)
RNase inhibitor
H2O
1 µl
0.5 µl
2.25 µl
* some buffer do not contain DTT, in this case add DTT to 10 mM final concentration
Mix gently by pipetting up and down, and incubate at 37 C for 1 hour.
Hydrolysis
Add 10 µl of 1 M NaOH and 10 µl of 0.5M EDTA pH8
Incubate at 65 C for 15 min
Add 25 µl 1M HEPES pH 7.4 to neutralize the reaction
Purification
Purify the amino-allyl cDNAs using the qiagen qiaqick kit, elute in two times 30 to 50 µl H2O
pH8
Speed vacuum to dry
Cy3 and Cy5 labeling
To the amino-allyl cDNAs (dry pellet), add 9 µl of 0.1M sodium bicarbonate.
Let the cDNAs resuspend for 15 min at RT.
Add 5 µl of Cy3 or Cy5, incubate at RT for one hour.
Add 4.5 µl 4M hydroxylamine, incubate at RT for 15 min.
Combine Cy3 and Cy5 cDNAs of the couple of samples to be hybridized together on the same
slide.
Clean up using the qiagen qiaquick PCR purification kit, elute two times, each time with 50 µl
H2O.
Add 3 µl polyA (10 mg/ml)
Speed vacuum to dry.
Hybridization
Resuspend the Cy3 or Cy5 labeled cDNAs in 40 to x µl of hybridization buffer (50% deoinized
formamide, 5XSSC, 0.1% SDS) (the volume added depends on the coverslip specificities).
Incubate for 2 min at 94 C, chill on ice few min, than spin the tubes.
Hybridize overnight at 42 C.
Post-Processing of the poly-lysine slides see the protocol below:
FGL Post Processing of Long Oligo Microarray Slides (Vivian Peng)
revised 7/21/2005
1. No hydration of spots is necessary.
2. Cross-link 200mJ
3. Block in BSA as per Erle Lab protocol. All stock reagents should be filtered.
Shake slides in 0.2% SDS
Wash in copious amounts of ddH2O
4. Put slides directly into prehybridization solution [0.1% SDS, 5X SSC, 1% BSA]
Pre-warm the BSA at 42oC before making the prehyb for 15 minutes.
5. Incubate at 42oC for 30-45 minutes.
6. Wash slides in many (about 5-6) changes of water.
7. Spin to dry, 1000rpm for 3 min or 500 rpm 5 mins.
Notes: Plunge slides gently 5-6 times whenever transferring into new solution.