Labeling Procedure For Microarray Analysis
Updated 2011
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I. General
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In today's experiment you will be making the cDNA probes for our microarrays. Each group will make a probe from WT RNA and a probe from the RNA from a mutant yeast strain. For making our labeled probe we will be using the Genisphere 3DNA Array 350 Detection Kit. The labeling procedure will occur in steps. The first step involves reverse transcribing the mRNA from each yeast strain into cDNA using the special primer provided. This primer is a poly TTTTT primer that has a special 3DNA cature sequence at the end of it.
Here is what the primers look like that you will use for this first step--
The Poly TT part of the the primer will bind to the polyA regions of the RNA and be used to make a copy of the RNA. The 3DNA capture sequence is a nucleic acid sequence that is different for each of the two dyes. There is one sequence for the red dye and one sequence for the green dye.
In a subsequent step we will mix our cDNAs with the two dyes called Cy3 (green) and Cy5 (red). The dyes are attached to an oligonucleotide with a sequence complimentary to the 3DNA capture sequence. These oligonucleotides will hybridize to your cDNAs' 3DNA capture sequences thus labeling them with a red or green dye depending on the primer you used. Below is a picture of what the probe will look like that you will use on the arrays. I will give you more information about this in class.
Cy-dye - 3' complimentary to capture sequence-5'
Remember to continue to use caution with your RNAs as we don't
want them to get degraded until they have been transcribed into cDNA.
Remember to use your best pipetting
skills.
1. Determine which product will be used (we will use our purity and gel data from the previous class) and labeled with the green dye and which will be labeled with the red dye.
2. Second perform all calculations and then set up RNA-RT primer mix for each RNA sample in a seperate labeled 0.5 ml RNase-free tube:
3. Mix the samples well by vortexing and spin to the bottom. Note: To spin these small tubes make sure you insert them inside a large tube first or they will fall through the holes in the centrifuge.
4. Heat to 75° C for 10 minutes in waterbath and place on ice immediatly after you take the tubes out of the bath. This denatures the RNA and primer to get rid of secondary structure. Make sure the temperature is not higher then this or you will destroy your RNA!
5. Snap cool on ice for 2 min.
6. In a seperate 0. 5 ml tube use careful pipetting to add each of the following ingrediants to make a master reaction mix. This reaction mix should be kept on ice at all times. The total volume of this solution is 12 ul.
7. Mix gently by pipetting(do not vortex) then spin the contents to the bottom of the tube. Add 6 ul of this solution to your RNA/primer mix from step 4 - mix gently). There should be 20 µl in the tube.
8. Incubate 42° C for 2 hours (note we usually do this only for 1 hour and 30 minutes) in a waterbath.
9. Stop the RT reactions by adding 3.5 µl of 0.5 M NaOH / 50 mM EDTA and incubating for 10 min at 65 degrees C. This denatures the RNA/DNA hybrids and degrades the RNA.
10. Cool the reactions on ice and neutralize with 5 µl of 1 M Tris-HCl, pH 7.5. (Total volume = 29.5 µl)
11. Combine your two RT products together into one of the two tubes. Rinse the tube that you took the first product out of with 73 ul of TE buffer pH = 8.O and put the rinse solution in the tube with the combined products. This should give you 130 ul of cDNAs.
In order to do the hybridization we need to get the cDNA into a much smaller volume to do this we will use YM30 microconcentrators.
1. To a YM30 microconcentrator into a 1.5 ml collection tube.
2. Add 100 ul TE buffer pH = 8.0 to the resivour to wash the membrane. Do not touch the membrane with a pippette tip.
3. Place the concentrator in a microfuge tube and spin for 3 minutes at 11K rpm (10-14K x g specified).
4. Discard the TE buffer.
5. Transfer the 130 ul of cDNA to the reservoir- do not touch the membrane with your pippette tip.
6. Spin the concentrator for 9 minutes at 11K rpm.
7. After the spin, remove the reservoir and add 5 ul of TE to Reservoir without touching the membrane. Tap sample to spread the TE accross the membrane.
8. Invert the reservoir over a fresh tube and spin for two minus at 14K rpm (top speed).
9. Discard the reservoir and measured eluate with a micropipette to find out how much you have. You should have between 3-10 ul. Add Nuclease Free Water (vial 10) to obtain a final volume of 10 ul. Freeze at -70 degrees Celcius.