Dahlquist:DNA Microarray Protocol

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(Day 3)
(Day 3)
Line 139: Line 139:
# Bring volume of aRNA to 5 μL using nuclease-free water.
# Bring volume of aRNA to 5 μL using nuclease-free water.
# Add 5 μL of 1:1 formaldehyde loading dye (Lonza) to each sample.
# Add 5 μL of 1:1 formaldehyde loading dye (Lonza) to each sample.
-
#* Remove aliquotted sample of 1 kb RNA markers from -80°C freezer and include in subsequent steps.
+
#* Remove aliquotted sample of 0.5-9 kb RNA markers (Lonza) from -80°C freezer (dye already added) and include in subsequent steps.
# Incubate 65°C for 15 minutes.
# Incubate 65°C for 15 minutes.
# Incubate on ice for 15 minutes.
# Incubate on ice for 15 minutes.
# In the meantime, place the pre-cast Reliant gel on the loading tray of the large horizontal gel box.
# In the meantime, place the pre-cast Reliant gel on the loading tray of the large horizontal gel box.
#* Make sure that it is not crooked.
#* Make sure that it is not crooked.
-
# Fill box to cover gel with 1X MOPS Running Buffer.
+
# Fill box to cover gel with 1X AccuGENE MOPS Running Buffer (Lonza).
 +
#* Dilute 100 mL 10X AccuGENE MOPS Running Buffer with 900 mL MilliQ water to make 1 L.
 +
#* Can be saved and re-used several times (stored at room temperature) for this type of gel.
# Run at 65 volts constant voltage for 2 hours.
# Run at 65 volts constant voltage for 2 hours.
# Remove gel from casting tray and place in small tupperware container.
# Remove gel from casting tray and place in small tupperware container.
Line 150: Line 152:
# Add 10 μL of 10,000X GelStar Nucleic Acid Gel Stain (Lonza).
# Add 10 μL of 10,000X GelStar Nucleic Acid Gel Stain (Lonza).
# Gently agitate for 30 minutes.
# Gently agitate for 30 minutes.
-
# Detect the RNA using UV transillumination at Kodak Gel Logic 100 station.
+
# Detect the RNA using UV transillumination with Kodak Gel Logic 100 station.
#* Amplified aRNA should appear as a smear from 250 to 5000 nt.  The average size of amino allyl aRNA should be approximately 1400 nt.
#* Amplified aRNA should appear as a smear from 250 to 5000 nt.  The average size of amino allyl aRNA should be approximately 1400 nt.
-
 
+
#* Mix together aRNA samples if multiple reactions were performed for the same total RNA sample and each passes quality control.  Re-read absorbance values and re-calculate concentration.
-
* Mix together aRNA samples if multiple reactions were performed for the same total RNA sample.
+
# Calculate the volume required for 20 μg of aRNA and aliquot into a fresh 1.5 mL microcentrifuge 
To be continued...
To be continued...

Revision as of 19:06, 6 August 2009

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Contents

Overview

This protocol describes all the steps necessary to perform aRNA synthesis, labeling, hybridization, and scanning of DNA microarrays for Saccharomyces cerevisiae, beginning with total RNA.

NOTE: This protocol is still under construction!

Materials

Supplies

NOTE: RNase-free supplies should be used whenever possible.

  • SealRite 1.5 mL Natural Microcentrifuge Tubes (free of detectable RNase, DNase, DNA & pyrogens; USA Scientific Catalog #1615-5500)
  • TipOne 101-1000 μL Filter Tips (sterile, free of detectable RNase, DNase, DNA & pyrogens; USA Scientific Catalog #1126-7810)
  • TipOne 1-200 μL Graduated Filter Tips (sterile, free of detectable RNase, DNase, DNA & pyrogens; USA Scientific Catalog #1120-8810)
  • TipOne 0.1-10 μL Filter Tips (sterile, free of detectable RNase, DNase, DNA & pyrogens; USA Scientific Catalog #1121-3810)
  • 50 ml conical screw cap tubes, copolymer, bulk, sterile (USA Scientific Catalog #1500-1211)
  • Powder-free latex exam gloves (USA Scientific Catalog #4900-2200, 4900-3300, 4900-4400)
  • multiwipes, small and large (Denville Scientific Catalog #C5709-1A, C5709-3A)
    • a.k.a. Kim wipes
    • Denville Scientific sells a generic version that is less expensive than the name brand
  • aluminum foil (from grocery store or other such supplier)
  • compressed air duster
    • VWR sells something called the "Whoosh Duster" (VWR Catalog #16650-027), but I've found that one purchased from an electronics store (less expensive) works fine (Kam D. Dahlquist 20:46, 15 August 2008 (EDT))
  • trUView™ Disposable Cuvettes (BioRad Catalog #1702510)
    • UV-transparant microcuvettes hold 50 μL total volume
    • can be cleaned and re-used indefinitely when you don't need to recover the sample from them
  • RNaseZap (Denville Scientific Catalog #D1180)
    • Actually an Applied Biosystems/Ambion product Catalog #AM9780, but it is less expensive if you buy it from Denville

Reagents

NOTE: RNAse-free reagents should be used whenever possible.

  • Amino Allyl MessageAmp™ II aRNA Amplification Kit (Applied Biosystems Catalog #AM1753)
  • CyDye Post-Labeling Reactive Dye Pack (GE Healthcare Life Sciences Catalog #RPN5661)
  • TE Buffer (USB Catalog #75893)
  • Nuclease-free water (non-DEPC treated)
    • Nuclease-free water is provided in the Amino Allyl MessageAmp™ II aRNA Amplification Kit and is sufficient for the μL quantities called for throughout the protocol; additional water can be purchased from Applied Biosystems/Ambion, but it is expensive.
    • DEPC is an oxidizer and should be fully removed from water used with DNA microarrays because the Cy5 dye is prone to degradation by oxidation.
    • For larger quantities of nuclease-free water (non-DEPC treated) that are needed to dilute 20X SSC for washes, for example, I use Water, ASTM Type II, non-sterile, Reagent Grade, ACS (VWR Catalog #RC91505) that can be purchased in 10 L or 20 L cubes. This water was recommended by Genisphere in the DyeSaver 2 protocol as being validated for use with microarrays and not containing components that will oxidize Cy5; Kam D. Dahlquist 20:24, 15 August 2008 (EDT))
  • RNA Fragmentation Reagents (Applied Biosystems Catalog #AM8740)
  • DIG Easy Hyb (Roche Applied Science Catalog #11796895001)
  • Fish sperm DNA Solution 10 mg/mL
    • Have used Ultra Pure Salmon Sperm DNA Solution from Invitrogen (Catalog #15632-011)
    • When that runs out will use DNA, MB-grade from fish sperm from Roche Applied Science (Catalog #11467140001) which is significantly less expensive
  • Oligo dA, 10-20mer, 1μg/mL (Invitrogen Catalog #POLYA.GF)
  • 100% ethanol (Sigma Catalog #E702-3)
    • for adding to wash buffers in Amino Allyl MessageAmp™ II aRNA Amplification Kit
  • 20X SSC (USB Catalog #19629)
    • for making post-hybridization wash buffers
  • 20% SDS (USB Catalog #75832)
    • for making post-hybridization wash buffers
  • Hybri-slips (Sigma Catalog #H0784)
    • pack of 100
    • inexpensive, disposable, easy-to-use, made out of plastic
    • L × W × thickness 60 mm × 24 mm × 0.25 mm
  • Yeast 6.4K Array (Y6.4K) (University Health Network Microarray Centre, Toronto)
  • Slide Coating Solution
    • "Home-brew" version of Genisphere DyeSaver 2
    • 2% PEG (MW cut-off 2000) in 1:1 solution of acetone and toluene
    • 200 mL needed to fill Coplin jar
    • Weigh 4.0 g of polyethylene glycol (Mn ca. 2000) and transfer to a dry glass reagent bottle. Add 100 mL of HPLC grade acetone (water content less than 0.5%) and close the bottle with a lid. Swirl the bottle gently to dissolve the solid completely. When a clear solution is formed add 100 mL of anhydrous toluene and mix the solution before using in the coating experiments. Note: do not use any plastic in this procedure. Store at room temperature in a fume hood.
    • Can be re-used

Equipment

  • Dedicated set of RNase-free pipetmen
  • Speedvac
  • Water bath set to 50°C
  • Water bath set to 37°C
  • Heat block set to 70°C
  • Microcentrifuge
  • Corning hybridization chambers
  • racks (for 1.5 mL microcentrifuge tubes)
  • racks (for 50 mL conical tubes)
  • ice bucket and ice
  • forceps
  • slide box
  • glass Coplin jar (to hold Slide Coating Solution)
  • UV/vis spectrophotometer

Procedure

General Notes

  • Use RNase-free reagents and supplies and maintain good RNase-free technique throughout.
  • At the beginning of each day's work, clean the bench top and pipetman shafts with RNaseZap.
  • Keep all samples on ice unless specifically noted otherwise.
  • Once the Cy3 and Cy5 dye packages have been opened, all procedures must be carried out in the dark.
  • Generally, we perform up to 10 reactions at one time.
  • Make sure to perform multiple reactions for the reference sample so that there is enough aRNA for each of the chips.
  • If using kit for the first time, add the appropriate amount of ethanol to the Wash Buffer.

Day 1

  1. Dry 1 μg of yeast total RNA to less than 10 μL in a SpeedVac.
    • 1 μg is the largest amount recommended for the kit.
    • RNA can be dried ahead of time and stored at -80°C.
  2. Reverse Transcription to Synthesize First Strand of cDNA: follow steps C1-C4 in protocol.
    • Can use heat block for 70°C incubation in step C2.
    • Use 42°C water bath for step C4.
    • Thaw reagents and make Master Mix for step D1 and immediately proceed to step D1 when 2 hours are up.
  3. Second Strand cDNA synthesis: perform steps D1-D2.
    • Use 16°C water bath in the cold room for step D2.
  4. Freeze samples at -20°C. Stopping point for Day 1.

Day 2

  1. cDNA Purification: Aliquot nuclease-free water needed for elution step (E4) plus some extra into a 1.5 mL microcentrifuge tube and pre-heat to 50°C in a water bath.
  2. Perform steps E1-E4.
  3. Proceed immediately to steps F1-F3 (In Vitro Transcription to Synthesize Amino Allyl-Modified aRNA.)
    • Perform step F2 in 37°C water bath for six hours. The protocol says to incubate for 4-14 hours. However, it is important to incubate the same amount of time for each experiment to reduce variability.
    • Freeze samples at -80°C. Stopping point for Day 2.

Day 3

  1. aRNA Purification: Aliquot nuclease-free water needed for elution step (G5) plus some extra into a 1.5 mL microcentrifuge tube and pre-heat to 50°C in a water bath.
  2. Perform steps G1-G5.
    • Do not vortex and do not centrifuge in steps G1 and G2 and proceed as quickly as possible to step G3, otherwise the aRNA will start precipitating and you will lose it.

Assess aRNA yield and quality through UV absorbance and agarose gel electrophoresis.

  1. Make a 1:50 dilution of each aRNA sample by diluting 2 μL of sample into 98 μL of TE Buffer.
  2. Read absorbance at 260 and 280 nm.
  3. Calculate the concentration of aRNA using the formula
A260 X dilution factor (50) X 40 μg/mL = concentration (μg/mL) (for 1 cm path length)
  • The 260/280 ratio should be between 1.7 and 2.1.
  • Calculate and aliquot the volume of aRNA required for 1 μg of aRNA into a fresh 1.5 mL microcentrifuge tube for each sample.
  • This could be a stopping point for Day 3, storing the samples at -80°C, but ideally, go on to the next step:

Agarose gel electrophoresis using Reliant RNA Gel System by Lonza

  1. Bring volume of aRNA to 5 μL using nuclease-free water.
  2. Add 5 μL of 1:1 formaldehyde loading dye (Lonza) to each sample.
    • Remove aliquotted sample of 0.5-9 kb RNA markers (Lonza) from -80°C freezer (dye already added) and include in subsequent steps.
  3. Incubate 65°C for 15 minutes.
  4. Incubate on ice for 15 minutes.
  5. In the meantime, place the pre-cast Reliant gel on the loading tray of the large horizontal gel box.
    • Make sure that it is not crooked.
  6. Fill box to cover gel with 1X AccuGENE MOPS Running Buffer (Lonza).
    • Dilute 100 mL 10X AccuGENE MOPS Running Buffer with 900 mL MilliQ water to make 1 L.
    • Can be saved and re-used several times (stored at room temperature) for this type of gel.
  7. Run at 65 volts constant voltage for 2 hours.
  8. Remove gel from casting tray and place in small tupperware container.
  9. Submerge gel, just to cover, with MilliQ water.
  10. Add 10 μL of 10,000X GelStar Nucleic Acid Gel Stain (Lonza).
  11. Gently agitate for 30 minutes.
  12. Detect the RNA using UV transillumination with Kodak Gel Logic 100 station.
    • Amplified aRNA should appear as a smear from 250 to 5000 nt. The average size of amino allyl aRNA should be approximately 1400 nt.
    • Mix together aRNA samples if multiple reactions were performed for the same total RNA sample and each passes quality control. Re-read absorbance values and re-calculate concentration.
  13. Calculate the volume required for 20 μg of aRNA and aliquot into a fresh 1.5 mL microcentrifuge

To be continued...

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
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References

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