Agilent Low RNA Input Fluorescent Linear Amplification Kit Protocol (5184-3523).
Fluorescent cRNA Synthesis Procedure
cDNA Synthesis From Total RNA
(Time required: ~3 hours)
1.
Add 50 to 500 ng total RNA in a volume of 10.3 µL
or less. The total concentration should be at least
5 ng/µL.
□
2.
Add 1.2 or 5 µL of T7 Promoter Primer (from kit).
□
3.
Use nuclease-free water to bring the total reaction
volume to 11.5 µL.
□
ng Sample for use
500
# Sample Name Conc. ng/µl µl Sample µl T7 primer
1
#DEEL/0!
2
#DEEL/0!
3
#DEEL/0!
4
#DEEL/0!
5
#DEEL/0!
6
#DEEL/0!
7
#DEEL/0!
8
#DEEL/0!
9
#DEEL/0!
10
#DEEL/0!
11
#DEEL/0!
12
#DEEL/0!
13
#DEEL/0!
14
#DEEL/0!
15
#DEEL/0!
16
#DEEL/0!
17
#DEEL/0!
18
#DEEL/0!
19
#DEEL/0!
20
#DEEL/0!
µl H2O
#DEEL/0!
#DEEL/0!
#DEEL/0!
#DEEL/0!
#DEEL/0!
#DEEL/0!
#DEEL/0!
#DEEL/0!
#DEEL/0!
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#DEEL/0!
#DEEL/0!
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#DEEL/0!
#DEEL/0!
#DEEL/0!
□
4.
Incubate the reaction at 65°C in a heating block for
10 minutes.
5.
6.
Place the reactions on ice and incubate for 5 minutes.
□
Immediately—prior to use, gently mix the following
□
components by pipetting, in the order indicated, at room temperature:
Agilent Low RNA Input Fluorescent Linear Amplification Kit Protocol
16-2-2016
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Be sure to always make a mix for 10% (round up to whole) reactions more or at least 1.
Number of reactions
2
Lot # kit:
cDNA Master Mix
5X First Strand Buffer
0.1 M DTT
10 mM dNTP mix
MMLV RT
RNaseOUT
Total
Volume (µl)
4
2
1
1
0.5
8.5
8
4
2
2
1
17
Pre-warm the 5X First Strand Buffer by incubating the
vial in a 65°C waterbath for 3-4 minutes.
Vortex briefly and spin the tube briefly in a microfuge.
7.
To each sample tube, add 8.5 µL cDNA Master Mix.
□
8.
Incubate samples at 40°C in a circulating water bath
for 2 hours.
□
9.
Move samples to a heating block or water bath set to
65°C and incubate for 15 minutes.
□
10.
Move samples to ice. Incubate on ice for 5 minutes.
□
11.
Spin samples briefly in a microcentrifuge.
□
Fluorescent cRNA Synthesis: in vitro transcription and incorporation of cyanine 3or cyanine 5-CTP
(Time required: 2.5 hours)
12.
Immediately, prior to use, gently mix the following
components by pipetting, in the order indicated, at
room temperature:
□
Be sure to always make a mix for 10% (round up to whole) reactions more or at least 1.
Agilent Low RNA Input Fluorescent Linear Amplification Kit Protocol
16-2-2016
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Number of reactions
Number of reactions Cy-3
Number of reactions Cy-5
Transcription Master Mix
Nuclease-free water
4X Transcription Buffer
0.1 M DTT
NTP Mix
50% PEG
RNaseOUT
Inorganic Pyrophosphatase
T7 RNA Polymerase
Total
2
1
1
Volume (µl)
15.3
20
6
8
6.4
0.5
0.6
0.8
57.6
Cy3 CTP
30.6
40
12
16
12.8
1
1.2
1.6
115.2
NEL580
Lot#
Cy5 CTP
NEL581
Lot#
57.6 Mix
2.4 Cy3 CTP
60 Total
57.6 Mix
2.4 Cy5 CTP
60 Total
Pre-warm the 50% PEG solution by incubating the vial
in a 40°C waterbath for 1 minute. Vortex briefly and
spin the tube briefly in a microfuge.
13.
To each sample tube, add 60.0 µL of Transcription
Master Mix. Gently mix by pipetting.
Agilent Low RNA Input Fluorescent Linear Amplification Kit Protocol
16-2-2016
□
3
#
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
14.
Name
Label with
Incubate samples in a circulating water bath at 40°C
for 2 hours.
□
Purification of Amplified cRNA (Machery-Nagel NucleoSpin RNA II kit)
15.
Add 20 µL of nuclease free-water to your cRNA
sample
□
16.
Add 350 µL of Buffer RA1 and vortex to mix.
□
17.
Add 350 µL of ethanol (70%) and mix
by vortexing.
□
18.
Transfer 800 µL of cRNA sample to an nucleospin RNA II
□
Agilent Low RNA Input Fluorescent Linear Amplification Kit Protocol
16-2-2016
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column.
Centrifuge the sample for 30 seconds at 11.000 RPM.
Place the column in a new collection tube.
□
19.
Add 200 µL of buffer RA2 to the nucleospin RNA II column.
Centrifuge the sample for 30 seconds at 11.000 RPM.
Place the column in a new collection tube.
20.
Add 600 µL of buffer RA3 to the nucleospin RNA II column.
□
Centrifuge the sample for 30 seconds at 11.000 RPM.
Discard flowtrough and place column back into the collecting tube.
21.
Add 250 µL of buffer RA3 to the nucleospin RNA II column.
Centrifuge the sample for 2 min at 13.000 RPM to dry the
membrane competely. Place the column into a nucleasefree
1,5 ml microcentrifuge tube.
□
22.
Elute the RNA in 60 µL nuclease free H2O and
centrifuge at 13.000 RPM for 1 min.
□
23.
Add the eluate from step 22 again directly onto the
nucleospin RNA II column.
Centrifuge at 13.000 RPM for 1 min.
□
1.
Perform wavescan on nanodrop to determine labelling efficiency and
amplification.
Save the report on disk.
2.
If requested evaluate labelled sample on an Agilent 2100 bioanalyzer.
Agilent Low RNA Input Fluorescent Linear Amplification Kit Protocol
16-2-2016
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Agilent Low RNA Input Fluorescent Linear Amplification Kit Protocol
16-2-2016
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Overview Concentrations and Hybridisation
#
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Name
Cy5
Label
Cy3
Conc. µg/ml
µg on array
Slide barcode
µl required
Batch number
A
B
C
D
E
F
G
H
Agilent Low RNA Input Fluorescent Linear Amplification Kit Protocol
16-2-2016
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Materiallist.
Materials for general purposes.











sterile nuclease-free reaction tubes (0,5 and 1,5 ml)
nuclease-free water
ice
Microcentrifuge
Incubator (waterbath or PCR machine 65ºC and 40ºC)
vortex
microplate centrifuge
Magnetic stir plate
Speedvac
Nanodrop
Bioanalyzer
cDNA Synthesis From Total RNA

50 to 500 ng total RNA
Low input fluorescent amplification kit (Agilent 5184-3523):
cDNA Master Mix






T7 Promoter Primer
5X First Strand Buffer
0,1 M DTT
10 mM dNTP mix
MMLV RT
RNaseOUT
Fluorescent cRNA Synthesis: in vitro transcription and incorporation of cyanine 3or cyanine 5-CTP
Low input fluorescent amplification kit (Agilent 5184-3523):
Transcription Master Mix







4X Transcription Buffer
0,1 M DTT
NTP Mix
50% PEG
RNaseOUT
Inorganic Pyrophosphatase
T7 RNA Polymerase


Cy3-CTP (Perkin-Elmer NEL580)
Cy5-CTP (Perkin-Elmer NEL581)
Agilent Low RNA Input Fluorescent Linear Amplification Kit Protocol
16-2-2016
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Purification of Amplified cRNA (RNeasy kit)




Buffer RLT
ethanol (96-100% purity)
RNeasy mini column
buffer RPE
Agilent Low RNA Input Fluorescent Linear Amplification Kit Protocol
16-2-2016
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