If we begin with certainties, we shall end in doubts; but if we begin with doubts,
and are patient in them, we shall end in certainties.
Francis Bacon

Day 1

***Prepare for the course by familiarizing yourself with the Background on Microarrays.***


Isolation of Total RNA from Arabidopsis thaliana

High quality RNA is critical to the success of microarray experiments. Degraded or contaminated RNA cannot be efficiently reverse transcribed or labeled and, therefore, will not produce good hybridization signals.

Genisphere 3DNA Array 900™ Expression Array Detection Kit

*Download the complete Array 900 Protocol in PDF format.
Do NOT print the entire manual--just read pages 2-15 and 20-21.

The 3DNA Array 900 kit is designed for arrays printed with PCR products (cDNA) or oligonucleotides.
First, total RNA is reverse transcribed using a special RT primer. Next, the cDNA and the fluorescent 3DNA reagent are hybridized to the array in succession; the 3DNA reagent contains a "capture sequence" that is complementary to a sequence on the 5' end of the RT primer.

Step 1: cDNA Synthesis from Total RNA (Reverse Transcription Reaction)

Remember, RNases ARE STILL EVERYWHERE!! Use the barrier pipet tips, wear gloves, and close the caps of all tubes.
  1. Set up a RNA-RT primer mix for EACH RNA sample (4 TOTAL = 2 control + 2 experimental) in 0.5 ml RNase-free tubes:

    1-5 l TOTAL RNA (0.5-2.5 g)
    1 l RT primer (use Cy3 for the control sample; use Cy5 for the experimental sample)
    nuclease-free water to a FINAL volume of 6 l

  2. Mix by gently tapping tube; pulse spin.
  3. Heat to 80°C for 5 minutes (use the thermal cycler block); IMMEDIATELY transfer to ICE for 2-3 minutes; pulse spin and return to ice.
  4. Prepare a Reaction Master Mix: Gently mix by tapping and pulse spin; keep on ice until ready to use.
  5. Add 4.5 l of the Reaction Master Mix to the 6 l RNA-RT primer mix (10.5 l FINAL volume).
  6. Mix by gently tapping tube; pulse spin.
  7. Incubate at 42°C for 2 hours (use the thermal cycler block).

    During this incubation, practice adding liquid to the coverslip with lifters:
    Use a KimWipe to remove dust from a clear glass slide; center a 25x60mm coverslip, LIFTERS-SIDE DOWN, on the slide; very SLOWLY and CAREFULLY pipet 60 l RO water at the edge of the coverslip; make sure that you use sterile WHITE tips, NOT the bulk YELLOW ones.
    NOTE: capillary action will pull the liquid under the coverslip; you should see it evenly spread until it reaches the other end of the coverslip.
    **The loading must be even and without any bubbles so that the entire array will be exposed to the labeled samples.
    {Loading the microarray is very similar to casting the IEF gel in Protein Lab--just on a much smaller scale.}

    We have cDNA! Breathe...Relax a little...but still wear gloves and keep tubes closed. NOW we can use regular autoclaved pipet tips.

  8. STOP the RT reaction by adding 1 l 1 M NaOH/100 mM EDTA.
  9. Incubate at 65°C for 10 minutes (use the thermal cycler block) to denature the DNA/RNA hybrids and degrade the RNA.
  10. Pulse spin the samples.
  11. NEUTRALIZE the reaction by adding 1.2 l 2 M Tris-HCl, pH 7.5 (12.7 l FINAL volume).
  12. Store cDNA synthesis reactions at -20°C until ready to proceed with cDNA Hybridization.

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 5 June 2002
Updated 7 March 2007