Scott E. Schaus
2/6/16
Protocol for Reverse Transcription with Amino-allyl dUTP and Coupling to
NHS-Cyanine Dye
Original procedure: www.microarrays.org/protocols.html
The following protocols were largely derived from the Cold Spring Harbor Microarray Course manual.
Contributors include: H.Bennett, J. DeRisi, V.Iyer, M. Diehn, A. Alizadeh, J.Lieb, and other members of the
Brown and DeRisi labs.
The following procedure is a modification of the protocol provided by Joe Derisi which was developed at Rosetta
Inpharmatics, Kirkland, WA
Reverse Transcription
Priming of RT Reaction
Oligo dT / pd N6 (5 µg / µl each)
Total volume of 2 µg Poly(A)-RNA
(0.7 – 4 µg Poly(A)-RNA may be used)
1 µl per reaction
14.5 µl in DEPC-H2O
Mix in RNAse free eppendorf tube
Incubate primer / Poly(A)-RNA mixture 70 ˚C, 10 min
Chill on ice 10 min
cDNA synthesis
5x buffer
50x aa-dUTP / dNTP
DTT (0.1 M)
H2O-(DEPC)
µl / rxn
6
0.8
3
3
µl / 4.5 rxn
27
3.6
13.5
13.5
12.8 µl / rxn
aliquot buffer mixture to each primer / mRNA mixture
add 2.5 µl Superscript RT to each reaction
Incubate 42 ˚C for 2 h minimum; 3 h maximum
Notes
1. The primer mixture is ordered as custom primers from Gibco/BRL; Oligo dT is a
mixture of dT 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer and 22-mer. The pd N6 is
ordered as NNNNNN from Gibco/BRL.
2. Amino-allyl dUTP is ordered from Sigma in 1 mg aliquots and dissolved in 20 µl H 2O(DEPC) and mixed with 30 µl of 100 mM dATP, dGTP, dCTP and 12 µl 100 mM dTTP.
The final ratio of aa-dUTP and dTTP is 3:2.
Hydrolysis
To each reaction add:
10 µl 1 N NaOH
10 µl 0.5 M EDTA
Mix well
Incubate 15 min at 65 ˚C
Neutralize each reaction by the addition of 25 µl 1 M HEPES pH 7.5 and mix well
Chill on ice until cleanup
May be stored at –20 ˚C for up to 2 weeks.
Scott E. Schaus
2/6/16
Cleanup
The transcipts obtained from the reverse transcription reaction must be purified and all of the Tris
removed before coupling to the NHS-cyanine dye.
Label each microcon-30 filter and weigh each filter to the mg (usually 635 mg)
Dilute each neutralized reaction to 500 µl with nuclease-free water (Ambion)
Add solution to tarred microcon-30
Spin 12000 rpm (10000 xg) for 9 min
Remove flow-thru
Add 470 µl nuclease-free water and spin 10000 xg 12 min
Remove flow-thru and repeat rinse
Spin microcon until weight difference is 18 mg (18 µl of solution remains in filter)
Collect at 4500 rcf for 5 min
The final volume collected should be 10 – 13 µl (the filter retains 5 – 7 µl)
Coupling
The amine of the amino-allyl uracil must be free-based before coupling to the N-hydroxy
succinamide ester of the cyanine dye
To the 10 µl of purified probe, add 1 µl of 1 M NaHCO3 pH 9, the final concentration
should be no less than 50 mM NaHCO3.
Add the basic solution of probe to an aliquot of NHS cyanine dye
Incubate in the dark for 1.25 h at rt
Note
The NHS-ester cyanine 3 and cyanine 5 dyes are purchased from Amersham as the
protein labeling dye pack. In each pack, there are 5 vials of dye aliquots. Each vial must be
aliquoted into 10 portions. The vial is dissolved in 20 µl DMSO (HPLC grade) and aliquoted into
10 DNAse-free eppendorf tubes. The aliquots are then concentrated on a Speed-Vac under low
heat. The dye aliquots are then stored 4 ˚C until use.
Purification
The probes are purified using Qiagen PCR Purification kit
Individually purify each Cy3 and Cy5 probe
Add 70 µl H2O to each labeling reaction
Add 500 µl PB buffer
Apply to Qiaquick column and spin 13000 rpm for 45 sec
Aspirate flow-thru
Add 700 µl PE buffer and spin 45 sec
Aspirate flow-thru and repeat
Aspirate flow-thru and spin 1 min 14000 rpm to dry column
Transfer to DNAse-free eppendorf tube
Add 30 µl EB buffer to filter and allow to stand 1 min rt
Spin 1 min 14000 rpm
Repeat elution step with another 30 µl EB buffer
Label and tare a microcon 30 filter
Combine the Cy3 and Cy5 probe for hybridization on filter
Spin microcon 30 filter 3.5 min 12000 rpm
Weigh microcon and continue to concentrate until 27 mg remain (20 - 22 µl recovery)
Collect probe in clean eppendorf tube 4500 rcf, 5 min
Scott E. Schaus
2/6/16
Hybridization Preparation
To the concentrated probe (20 - 22 µl) add:
3 µl 20X SSC
1.5 µl poly(A) (10 µg / µl)
0.48 µl 1 M HEPES pH 7.0
Wet Millipore 0.45 µm filter with 10 µl H2O
Spin 12000 rpm 1 min
Aspirate flow-thru
Apply probe to filter as a drop
Spin 12000 rpm 1 min
Remove filter
Add 0.45 µl 10% SDS
Set up slide in Telechem hybridization chamber
Clean Lifterslip with EtOH and Kimwipes
Place Lifterslip on the array being sure the white strips are down on the glass
Hybridization
Heat probe 2 min 100 ˚C
Allow probe to cool 10 min rt on bench top
Probe volume to be applied should be 23 µl
Slowly inject probe at one corner of the lifter slip and continue to apply the probe to the
corners being sure not to leave any air bubbles under the lifter slip.
Add 40 µl 3X SSC to each of the wells of the hybridization chamber
Seal hybridization chamber and place in 60 ˚C water bath, cautious to keep chamber
level during hybridization.
Allow hybridization to go 12 – 15 h, 60 ˚C