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The protocol for generating labeled cdna probes from total rna in yeast for use in microarray experiments. The procedure involves reverse transcription using special primers and fluorescent dyes, handling light-sensitive reagents, and concentrating the cdna using microcon ym-30 filter devices.
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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”
that is complementary to a sequence on the 5’ end of the RT primer. The signal from these special primers does not depend on the length or the base composition of the transcript, each cDNA is labeled by one 3DNA primer. RNA is extremely sensitive to degradation by RNases. How carefully you handle your samples and transfer solutions will have a huge impact on the quality of your microarray data.
Keep reagents on ice Always close pipet tip boxes Dispose of tips in waste containers, not on the bench Never use tips more than once!
Since microarrays and RNA preparations vary in quality, the exact amount of RNA required for an experiment will typically range from 1-5 μg of Yeast total RNA. (10 ul of our samples is approximately 5 μg of Total RNA.)
Add 22 ul of 1X TE Buffer to your Cy3/Cy5 “mixed” cDNA (Final Volume 50 μl) Proceed to Step 2: Concentration of cDNA.
The cDNA must be concentrated before it can be used in the hybridization mix. Although ethanol precipitation is a traditional method for nucleic acid concentration, this method may lead to variable results due to partial or complete loss of the pelleted cDNA or incomplete re- solubilization of the precipitated cDNA. Microcon concentration is an alternative method that may offer better performance characteristics. Note: Evaporative drying of the cDNA is NOT recommended, as a dried sample may result in lower signal and/or higher background on the array. Millipore Microcon YM-30 Centrifugal Filter Devices cDNA samples may be concentrated using the Millipore Microcon YM-30 Centrifugal Filter Devices (30,000 molecular weight cutoff, Millipore cat. no. 42409). These devices are capable of reducing the volume of the cDNA synthesis reaction to 3-10μl in as little as 8-10 minutes. The procedure below reiterates the manufacturer’s directions with minor adaptations for the 3DNA Array 350 Kit. Important: When using the Microcon YM-30 column, evaluate centrifuge time and speed to yield final volumes of 3-10μl. A fixed angle rotor tabletop centrifuge (microfuge) capable of 10-14,000g should be used.