Cell & Molecular Biology Laboratory
BIOL 406 cDNA Probe generation for Yeast Microarrays
Labeled cDNA Probe Generation from Yeast RNA
Objectives:
1. Use Total Yeast RNA to artificially synthesize labeled cDNAs for hybridization in microarrays.
2. Understand the function of Reverse Transcriptase, Primers & use of Fluorescent tags
3. Handle light sensitive reagents properly
4. Understand how the method of labeling cDNAs can affect final signal observed in microarrays
5. Work with limited reagents and small volumes using extreme caution
6. Understand the steps required in experimental design and planning
READ THIS PROTOCOL CAREFULLY. MAKE SURE YOU ARE
PREPARED TO ASK ANY TECHNICAL OR THEORETICAL QUESTIONS
BEFORE WE BEGIN THIS EXPERIMENT. IF IN DOUBT ABOUT
ANYTHING, ASK!
Probe Generation from Total Yeast RNA
We will use the 3DNA Kit from Genisphere to synthesize our labeled cDNAs. To measure
changes in the mRNA levels in Yeast during the diauxic shift, we need to have a baseline level of
gene activity (i.e. mRNA levels). The initial time point (9 hours) with excess glucose in the media
will be our reference level of mRNAs. We will prepare labeled cDNA by reverse transcription
using special primers that will bind the fluorescent dyes Cy3(green) or Cy5(red). The cDNAs will
be hybridized to the microarrays in next week’s lab.
To ensure that we measure accurate changes in mRNA levels, we will label cDNA from the initial
time point (reference) with Cy3 (green), and cells at each successive time point (13, 17 and 21
Hours) with Cy5 (red). Cy5 labeled DNA from each time point will then be mixed with an aliquot of
the Cy3-labeled “reference” cDNA. In this experimental design, the relative fluorescence intensity
measured for the Cy3 and Cy5 fluors at each array element provides a reliable measure of the
relative abundance of the corresponding mRNA in the two cell populations (i.e. initial vs. later
time points). When there are equal amounts of mRNA present in two cell populations, equal
amounts of Cy3 and Cy5 will bind to the microarray producing a ‘yellow’ image. If some genes are
induced over the time course (higher mRNA levels), then those spots will appear red in the
microarray. Conversely, if a gene is repressed (lower mRNA level) the spot will appear green on
the microarray. In this way, we can monitor all 6000 genes in yeast on a single microarray slide.
The 3DNA Array 350 kit is easy to use and is designed for use with microarrays like ours. The
first step is to reverse transcribe the RNA using a deoxynucleotide triphosphate mix (dATP,
dCTP, dGTP & dTTP), Reverse Transcriptase and special RT dT primers. (Why don’t we just
label the RNA instead?) The dT primer (i.e. poly T) binds to the poly A tail of mRNAs via its 3’
end, the reverse transcriptase enzyme binds to this double stranded region and transcribes a
cDNA. The primers also have special ‘capture’ sequences that are used to bind to fluorescent
dyes on their 5’ ends, (either Cy3 or Cy5). The dyes are attached to the primers after the cDNAs
are synthesized. Many other kits use fluorescently tagged nucleotides, which can result in less
uniformly labeled cDNAs because of sequence dependent incorporation (some sequences are
transcribed more or less efficiently, leading to a bias in the data). The ‘capture’ sequences of the
3DNA primers avoids the problem of sequence dependent incorporation of the dyes. Finally,
we’ll hybridize the cDNA and the fluorescent 3DNA reagents to the microarray in succession. The
fluorescent 3DNA reagent will hybridize to the cDNA because it includes a “capture sequence”
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