Ambion MessageAmp II Amino Allyl

and Cy Dye Coupling for Oligo Arrays

Kate Rubins/Brown Lab – 04/14/2006 (update 05/25/2006)

Using Ambion MessageAmpII Amino Allyl with Cy Dyes - Part Number 1797 http://www.ambion.com/catalog/CatNum.php?1797

 

Note: You must use amino allyl incorporation during the in vitro transcription to ensure correct directionality of the probe for oligo arrays.

Before Using Kit for First Time: Prepare wash buffer by adding 32 ml ACS grade 100% ethanol to the bottle. Mix well and indicate on the bottle that ethanol has been added.

Day 1

First Strand cDNA Synthesis

  1. We recommend at least 500ng Total RNA input if possible, up to 1µg. Less than 200ng may not yield enough aRNA. In PCR reaction tube, mix:

Amount

Reagent

100ng-1µg

Total RNA

1µl

T7 Oligo (dT) primer

1µl

Nuclease free water

Bring to 12µl

Total Volume

  1. 70°C for 10 minutes in a thermocycler with lid on, but not clamped down.
  2. Remove RNA samples from 70°C and centrifuge briefly. Place on ice.
  3. Prepare in a Master Mix and keep at room temperature (with 5% overage for pipetting error):

Amount

Reagent

2µl

10X First Strand Buffer

4µl

dNTP Mix

1µl

Ribonuclease Inhibitor

1µl

Array Script

8µl

Total Volume

  1. Gently pipette Master Mix or flick to mix, and then centrifuge briefly.
  2. Transfer 8µl of above Master Mix to each RNA sample, mix by pipetting.
  3. Gently pipette the reactions or flick to mix, and then centrifuge briefly.
  4. 42°C for 2 hours in thermocycler with lid on, but not clamped down.
  5. Centrifuge briefly, place on ice, and immediately proceed to next step of dsDNA synthesis. 

Second Strand Synthesis

  1. Prepare the following Master Mix on ice (with 5% overage for pipetting error):

Amount

Reagent

63µl

Nuclease free water

10µl

10X Second Strand Buffer

4µl

dNTP Mix

2µl

DNA Polymerase

1µl

RNase H

80µl

Total volume

  1. Gently pipette Master Mix or flick to mix, and then centrifuge briefly.
  2. Add 80µl to each tube.
  3. Gently pipette the reactions or flick to mix, and then centrifuge briefly.
  4. 16°C for 2 hours in thermocycler with lid completely off. Do not place the tubes in the thermal cycler until it has cooled to 16°C.

Note: Subjecting reactions to temperatures of less than 16°C will reduce aRNA yield.

  1. After 2 hour incubation, proceed to cDNA purification (or place reactions on ice for less than 1 hour) or freeze immediately at –20°C.

 

Double Stranded cDNA Cleanup

  1. Preheat the 5mL bottle of water at 50 to 55°C for at least 10 minutes during the previous 2hr incubation. Do not heat to >58°C.
  2. Transfer 100µl reaction from strip-8 PCR tubes into 1.5ml Eppendorf tubes, keeping on ice.
  3. Make sure there are no crystals in the bottom of the cDNA binding buffer. Warm it, if there are crystals.
  4. Check that the filter in the cDNA filter cartridge is flush against the bottom. Push down with a pipette tip if necessary.
  5. Add 250µl of cDNA binding buffer to each cDNA sample, and mix by pipetting.
  6. Add the mix to the center of the cDNA filter cartridge.
  7. Centrifuge for 1 minute at 10,000 x g.
  8. Discard flow-through but re-use Wash Tube.
  9. Add 500µl of cDNA wash buffer (ensure that ethanol has been added to bottle)
  10. Centrifuge for 1 minute at 10,000 x g.
  11. Discard flow-through but re-use Wash Tube.
  12. Centrifuge again for 1 minute at 10,000 x g to remove trace amounts of ethanol.
  13. Transfer filter cartridge to cDNA Elution Tube (provided).
  14. Add 9µl of preheated water to the center of the column.
  15. Incubate at room temperature for 2 minutes.
  16. Centrifuge for 1.5 minute at 10,000 x g.
  17. Repeat elution with a second 9µl of preheated water.
  18. Centrifuge for 1.5 minute at 10,000 x g.
  19. Proceed to IVT, or freeze cDNA at -20°C. 

In Vitro Transcription

  1. Prepare the following Master Mix at room temperature:

Amount

Reagent

3µl

Amino-allyl UTP (50mM)

12µl

ATP,CTP,GTP mix (25mM)

3µl

UTP (50mM)

4µl

T7 10X Reaction Buffer

4µl

T7 Enzyme Mix

26µl

Total Volume

  1. Gently pipette Master Mix or flick to mix, and then centrifuge briefly.
  2. Add 26µl to each tube.
  3. Gently pipette the reactions or flick to mix, and then centrifuge briefly.
  4. Incubate at 37°C for 14 hours in a hybridization oven. Use an internal thermometer to verify temperature.

Day 2

aRNA Purification

  1. Preheat the 5mL bottle of water at 50-60oC for at least 10 minutes.
  2. Add 60µl Nuclease free water to each aRNA sample to bring the final volume to 100µl.
  3. Mix by pipetting or gentle vortexing.
  4. Add 350µl of aRNA Binding Buffer to each aRNA sample.
  5. Add 250µl of ACS grade 95% ethanol to each sample.
  6. Mix by pipetting gently 3 times.

Note: Do NOT vortex and do NOT centrifuge. Proceed IMMEDIATELY to the next step.

  1. Apply entire sample to the equilibrated aRNA filter cartridge.
  2. Centrifuge for 1 minute at 10,000 x g.
  3. Discard flow-through but re-use tube.
  4. Add 650µl aRNA Wash Buffer to each filter.
  5. Centrifuge for 1 minute at 10,000 x g.
  6. Discard flow-through but re-use tube.
  7. Centrifuge again for 1 minute at 10,000 x g to remove trace amounts of ethanol.
  8. Transfer cartridge to a fresh aRNA Collection Tube.
  9. Elute aRNA by adding 100µl preheated (50 to 60°C) water to center of column.
  10. Incubate at room temperature for 2 minutes.
  11. Centrifuge for ~1.5 minute at 10,000 x g.
  12. Store aRNA at –80oC.

 

Gel/Spec

  1. Check RNA concentration and quality by measuring OD260 and OD260/280 using 1 on the NanoDrop spec.
  2. Optional — Check RNA quality by running a gel: Dilute 1µl sample into 2µl Sigma RNA Loading Buffer.
  3. Incubate at 65°C for 10 minutes. Place on ice immediately after incubation.
  4. Run on a 1.25% agarose gel (2.5g agarose in 200mL 1X MOPS) in 1X MOPS buffer for 45 minutes to 1.5hr at 165V.

 

Amino Allyl Coupling

  1. Add 11µl high quality DMSO to one tube of Cy3 or Cy5 dye supplied with kit.
  2. Vortex to mix thoroughly. Keep dye in the dark until ready to use (do not prepare dye >1hr before using). Make sure than no water gets in the dye/DMSO mix at any point.
  3. Add 5-20µg of amino allyl aRNA in a strip-8 PCR tube and Speedvac until completely dry. Remove tube from Speedvac as soon as it is dry - do not overdry! We recommend 10µg input into the coupling reaction, particularly for clinical samples or RNA that might be slightly degraded.  If low signal is a problem, increase amount of aRNA into the coupling reaction.
  4. Add 9µl coupling buffer and re-suspend thoroughly by vortexing gently.
  5. Add 11µl of prepared DMSA/Cy dye. Mix well by vortexing gently.
  6. Incubate 30 minutes at room temperature in the dark (cover in tinfoil, or put in a closed drawer).
  7. Add 4.5µl Hydroxlyamine to quench the reaction and mix well by vortexing gently.
  8. Incubate 15 minutes at room temperature in the dark (cover in tinfoil, or put in a closed drawer).
  9. Add 5.5µl of nuclease-free H20 to each sample to bring the volume to 30µl. 

Labeled aRNA Purification

  1. Preheat the 5mL bottle of water at 50-60°C for at least 10 minutes.
  2. Add 105µl of aRNA Binding Buffer to each aRNA sample.
  3. Add 75µl of ACS grade 95% ethanol to each sample.
  4. Mix by pipetting gently 3 times.

Note: Do NOT vortex and do NOT centrifuge. Proceed IMMEDIATELY to the next step.

  1. Apply entire sample to the equilibrated Labeled aRNA filter cartridge.
  2. Centrifuge for 1 minute at 10,000 x g.
  3. Discard flow-through but re-use tube.
  4. Add 500µl aRNA Wash Buffer to each filter.
  5. Centrifuge for 1 minute at 10,000 x g.
  6. Discard flow-through but re-use tube.
  7. Centrifuge again for 1 minute at 10,000 x g to remove trace amounts of ethanol.
  8. Transfer cartridge to a fresh Labeled aRNA Elution Tube.
  9. Add 10µl of preheated water to the center of the column.
  10. Incubate at room temperature for 2 minutes.
  11. Centrifuge for 1.5 minute at 10,000 x g.
  12. Repeat elution with a second 10µl of preheated water.
  13. Centrifuge for 1.5 minute at 10,000 x g.

 

Fragmentation/Spec

  1. Check RNA concentration and dye incorporation by measuring 1µl on the NanoDrop.
  2. Transfer the Cy5 samples to a 0.5ml tube.
  3. Pool the Cy3 samples in a 1.5ml tube and determine the total volume. Divide the total volume by the number of arrays to be hybridized. Transfer this amount of Cy3 sample into each Cy5 sample. Check that the final volume of Cy3 + Cy5 is equal to the required probe volume.
  4. Use RNA fragmentation reagents from Ambion (Catalog # 8740)
  5. Bring the labeled aRNA sample up to 27ul with Nuclease-free water from Ambion
  6. Add 3ul of 10x Fragmentation buffer to each sample.
  7. Incubate at 70°C for 15 minutes in a heating block, thermocycler or water bath.
  8. Add 3ul of Stop Solution (200mM EDTA, pH 8.0). Place samples on ice until ready to use or store them at –80°C.

 

Probe Preparation

  1. Concentrate Cot-1 DNA from 1µg/µl to 10µg/µl in a Speedvac. Check that the Yeast tRNA and PolyA RNA are at 10µg/µl.
  2. Make Cot, PolyA and yeast tRNA mix:

 

µl

10 µg/µl Cot1 human DNA (Gibco-BRL)

2

10 µg/µl polyA RNA (Sigma, #P9403)

2

10 µg/µl tRNA (Gibco-BRL, #15401-011)

2

Total volume

6

  1. Add Cot, Poly A and yeast tRNA mix, then the 20X SSC, then 10% SDS to the combined Cy 5 and Cy3 probe.

Note: Thin coverslips are not recommended, due to coverslip bending issues.

Cover Slip Size (mm)

Total Hyb Volume (µl)

Probe Vol (µl)

Cot, PolyA, tRNA Mix

20x SSC (µl)

10% SDS (µl)

1M (ph 7.0) HEPES

22x60 regular thin microscope slide coverslips (i.e. VWR)

35

28

6

5.95

1.05

0.84

22x60 Erie M-series thick Lifter Slips

55

36

6

9.35

1.65

1.32

 

 

Note: Avoid introducing bubbles and never vortex after adding SDS.

Note: HEPES is recommended for all probes

 

  1. Denature probe by heating for 2 minutes at 100°C.
  2. Spin at 14,000 RPM for 5 to 10 minutes.