M. Fero. v.4
A. 1st Strand Synthesis
1. In a 1.5 mL tube add:
RNA (30 µg total or 2 µg mRNA)
1 µL (5 µg/µL) Oligo dT
q.s. 25 µL H2O
2. Incubate 70ºC, 10 min, then place on ice for 10 min.
3. Meanwhile, prepare RT Master Mix (per sample):
2 µL Superscript II
8 µL 5x 1st strand buffer
0.4 µL 1 M DTT
0.8 µL 50x dNTP mix
4 µL 10x AA-dUTP (optional: spike with 1 µL 32P-dCTP)
5. Add 15 µL of RT Master Mix to each sample.
4. Incubate 42ºC, 2 hrs.
B. RNA Hydrolysis
1. To each sample add:
12.5 1N NaOH
5 µL 0.5 M EDTA
2. Incubate at 65ºC, 15 min.
3. Neutralize each sample with:
C. Cleanup on GFX columns
1. Turn on Speed Vac refrigerators.
2. Add 0.5 mL GFX capture buffer. (optional: save 1 uL for 32P counts)
3. Load on GFX column. Spin 1 min. Discard flow-through.
4. Add 0.5 mL GFX wash buffer. Spin. Discard flow-through.
5. Elute in amber 1.5 mL tube:
D. Coupling to amino-reactive Cy dyes (e.g. Cy5 for sample, Cy3 for control)
1. Resuspend in 4.5 µL H2O
2. To a Cy-dye aliquot add: 2.25 µL 0.2 M NaHCO3
3. Quickly combine dye and DNA.
4. Incubate at r.t. in dark for 1 hr.
5. Quench by adding 4.5 µL 4 M NH2OH
6. Incubate at r.t. in dark for 15 min.
E. Cleanup in QiaQuick columns
1. Combine Cy5 and Cy3 samples (experiment and control).
2. Add: 70 µL H2O, 500 µL Buffer PB
3. Apply to a QiaQuick column. Spin 13K g for 30". Discard flow-through.
4. Add 750 µL Buffer PE. Spin. Discard flow through.
5. Repeat wash with PE.
6. Spin 1 min. to dry column.
7. Elute: Add 30 µL buffer EB, incubate 1 min. Spin 1 min. into a fresh amber tube.
8. Repeat elution 1x.
9. Speed-vac to dry down.
F. Preparation of Hybridization Solution
1. To each sample/control combination add:
Spin at 12,000 g x5 min.
3. Store at -20ºC in the dark.
Amino-Allyl
cDNA Labeling
MaterialsRNA sample 30 µg Total or 2
µg mRNA
Oligo dT (dT18VN) 5 µg/µL, 34.8 (µg/mL)/A260
Superscript II, 5x 1st strand buffer (Invitrogen)
1 M DTT (Sigma, 43816, $16.80/10 mL)
50x dNTP Mix (dATP, dCTP, dGTP @25 mM, TTP=15mM)
10x AA-dUTP, 2 mM (MW = 523 g/mol), (Sigma A0410, $193.50/1mg - dissolve in 9.5 mL H2O)
1 N NaOH, 0.5 M EDTA, 2 M HEPES
0.2 M NaHCO3 (bicarbonate), 4 M NH2OH
GFX spin columns
QiaQuick columns
PolyA (10 mg/mL) - Sterile filtered (0.45 µm)
20x SSC (175.5 mg/mL NaCl, 88.3 mg/mL NaCitrate)
Cy3 and Cy5 Mono-reactive dye (Amersham PA23001 and PA25001):
To 1 tube dye add 36 µL anyhdrous DMSO and divide into 16x 2µL aliquots
Anhydrous DMSO: DMSO + Molecular Sieves (Sigma #M0133)
Oligo dT (dT18VN) 5 µg/µL, 34.8 (µg/mL)/A260
Superscript II, 5x 1st strand buffer (Invitrogen)
1 M DTT (Sigma, 43816, $16.80/10 mL)
50x dNTP Mix (dATP, dCTP, dGTP @25 mM, TTP=15mM)
10x AA-dUTP, 2 mM (MW = 523 g/mol), (Sigma A0410, $193.50/1mg - dissolve in 9.5 mL H2O)
1 N NaOH, 0.5 M EDTA, 2 M HEPES
0.2 M NaHCO3 (bicarbonate), 4 M NH2OH
GFX spin columns
QiaQuick columns
PolyA (10 mg/mL) - Sterile filtered (0.45 µm)
20x SSC (175.5 mg/mL NaCl, 88.3 mg/mL NaCitrate)
Cy3 and Cy5 Mono-reactive dye (Amersham PA23001 and PA25001):
To 1 tube dye add 36 µL anyhdrous DMSO and divide into 16x 2µL aliquots
Anhydrous DMSO: DMSO + Molecular Sieves (Sigma #M0133)
Notes
This procedure produces 1st strand cDNA with an anchored oligo dT
primer and AA-UTP. It then couples couples amino reactive
Cy-dyes
to the AA-UTP. For spotted arrays control RNA should also be
labeled using the opposing dye.
A. 1st Strand Synthesis
1. In a 1.5 mL tube add:
RNA (30 µg total or 2 µg mRNA)
1 µL (5 µg/µL) Oligo dT
q.s. 25 µL H2O
2. Incubate 70ºC, 10 min, then place on ice for 10 min.
3. Meanwhile, prepare RT Master Mix (per sample):
2 µL Superscript II
8 µL 5x 1st strand buffer
0.4 µL 1 M DTT
0.8 µL 50x dNTP mix
4 µL 10x AA-dUTP (optional: spike with 1 µL 32P-dCTP)
5. Add 15 µL of RT Master Mix to each sample.
4. Incubate 42ºC, 2 hrs.
B. RNA Hydrolysis
1. To each sample add:
12.5 1N NaOH
5 µL 0.5 M EDTA
2. Incubate at 65ºC, 15 min.
3. Neutralize each sample with:
20 µL 2M HEPES
O.K. to store o.n. at 4ºC.
O.K. to store o.n. at 4ºC.
C. Cleanup on GFX columns
1. Turn on Speed Vac refrigerators.
2. Add 0.5 mL GFX capture buffer. (optional: save 1 uL for 32P counts)
3. Load on GFX column. Spin 1 min. Discard flow-through.
4. Add 0.5 mL GFX wash buffer. Spin. Discard flow-through.
5. Elute in amber 1.5 mL tube:
Add 50 µL H2O.
Incubate 1 min. Spin 1 min.
Repeat elution a 2nd time into the same tube. (Optional: save aliquot to measure 32P counts)
7. Dry in speed-vac.Repeat elution a 2nd time into the same tube. (Optional: save aliquot to measure 32P counts)
D. Coupling to amino-reactive Cy dyes (e.g. Cy5 for sample, Cy3 for control)
1. Resuspend in 4.5 µL H2O
2. To a Cy-dye aliquot add: 2.25 µL 0.2 M NaHCO3
3. Quickly combine dye and DNA.
4. Incubate at r.t. in dark for 1 hr.
5. Quench by adding 4.5 µL 4 M NH2OH
6. Incubate at r.t. in dark for 15 min.
E. Cleanup in QiaQuick columns
1. Combine Cy5 and Cy3 samples (experiment and control).
2. Add: 70 µL H2O, 500 µL Buffer PB
3. Apply to a QiaQuick column. Spin 13K g for 30". Discard flow-through.
4. Add 750 µL Buffer PE. Spin. Discard flow through.
5. Repeat wash with PE.
6. Spin 1 min. to dry column.
7. Elute: Add 30 µL buffer EB, incubate 1 min. Spin 1 min. into a fresh amber tube.
8. Repeat elution 1x.
9. Speed-vac to dry down.
F. Preparation of Hybridization Solution
1. To each sample/control combination add:
27.8 µL H2O
5.4 µL 20x SSC
2.8 µL polyA (10 mg/mL)
2. Load the DNA mixture on a 0.5 µm Millipore
spin column5.4 µL 20x SSC
2.8 µL polyA (10 mg/mL)
Spin at 12,000 g x5 min.
