Cell & Molecular Biology Laboratory
BIOL 406
Hybridization of cDNAS to Yeast Microarrays
1
Hybridization of Yeast cDNAs to Microarrays
Objectives:
1. Use Yeast cDNAs & fluorescent reagents for hybridization in microarrays.
2. Understand the function & 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!
Hybridization of tagged cDNA to Microarrays
We will use the 3DNA Kit from Genisphere to hybridize our cDNAs to the microarrays, glass
slides spotted with all the genes found in yeast. Once cDNAs are bound to the slide, we’ll
hybridize the fluorescent tags Cy3 and Cy5 using the capture sequences introduced by the
special 3DNA primers. 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 is our reference level of the mRNAs. The last step in the
previous lab was to mix the reference cDNA (Time Point 1 at 9 Hours growth) with cDNA from the
later time points (13, 17 or 21 Hours). The ratio of these two cDNAs represents the changing rna
expression levels during the diauxic shift in yeast.
The slides are prepared by several wash steps before and after hybridization, care must be taken
not to contaminate your cDNA sample or the surface of the microarray. Hybridization is done in
two distinct steps in this lab. First the cDNA is bound to the microarray. The cDNAs will bind to
complementary sequences (i.e. yeast genes) that have been spotted at a particular physical
location on the slide (microarray). The cDNAs are incubated for at least 16 hours @ 50ºC to allow
specific binding to the microarray. (This step was completed for you before today’s lab) Today’s
lab begins with the second step in the hybridization. This step involves binding the 3DNA capture
reagents to the cDNAs on the slide. We are employing two different fluorescent dyes, Cy3 (green)
& Cy5 (red), which will hybridize to their respective capture sequences on the cDNAs. The
different dyes provide a two channel signal that can be represented as increasing (ie red) or
decreasing (ie green) images for analysis. Each 3DNA molecule contains ~375 fluorescent
molecules providing a uniformly strong signal. Special care must be used when working with the
dyes as they are susceptible to oxidation and photobleaching, which can reduce or destroy the
signal intensity.
Cell & Molecular Biology Laboratory
BIOL 406
Hybridization of cDNAS to Yeast Microarrays
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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.
Clear your bench of all but the bare essentials.
Wear gloves (at all times!!) and wash down your entire area & pipettors with
'RNase ZAP' (special detergent that helps control RNases a bit.)
All microfuge tubes and pipet tips have only been touched with gloves and have
been sterilized extensively.
Don’t use kimwipes (or anything containing lint) on microarray slides
Cell & Molecular Biology Laboratory
BIOL 406
Hybridization of cDNAS to Yeast Microarrays
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Materials:
(3DNA Array 350 kit Genisphere)
Vial 1 Cy3 Fluor 546 (red cap) or Cy5 Fluor 647 (blue cap) 3DNA Array 350 Capture Reagent
Vial 7 2X Formamide-Based Hybridization Buffer
Vial 8 Anti-Fade Reagent
Vial 9 LNA TM dT Blocker
Vial 10 Nuclease Free Water
Store Vials 1-12 at –20°C in the dark. Vial 1 may be kept at 4ºC for short-term storage (∼1
week).
Instructions for Handling Kit Contents
Vial 1: Dyes are light sensitive, keep covered at all times using aluminum foil
Vials 8, 9, and 10: Thaw at room temperature, vortex, and briefly microfuge.
Reducing Degradation of Cy5 Fluor 647
Cy5 Fluor 647 dye performance may be affected by a variety of factors that are particularly
prevalent during the summer months. Exposure of the Cy5 Fluor 647 dye solutions and the
hybridized arrays to light and to oxidative environments may cause rapid fading of the Cy5 Fluor
647 dye, regardless of the labeling system used. Limiting or controlling the exposure of the arrays
to these environments has been shown to significantly reduce Cy5 Fluor 647 fading. Below are
recommendations for reducing the degradation of Cy5 Fluor 647 when performing microarray
experiments:
1.
2.
3.
4.
5.
Always keep solutions and arrays containing Cy5 Fluor 647 away from light,
particularly sunlight. Cy5 Fluor 647 will photo-bleach when exposed to light,
including normal fluorescent lighting.
Protect the hybridized, dried array from contact with air, particularly on hot and sunny
days. Ambient ozone levels resulting from summertime air pollution can cause oxidative
degradation Cy5 Fluor 647. Keeping the Cy5 Fluor 647-containing arrays in an inert
atmosphere (nitrogen) in a small container (50mL tube) can significantly delay fading
of the Cy5 Fluor 647. Adding small quantities of dithiothreotol (DTT) or beta mercaptoethanol (BME) to the bottom of the tube to further promote a reducing micro-environment.
Be certain to avoid contact of the array with these chemicals. If possible, do not place the
arrays or the array scanner near high ozone-generating equipment, such as a freezer or
laser printer.
Use the Anti-Fade Reagent (provided with the 3DNA kits) in the hybridization solution
containing Cy5 Fluor 647 Capture Reagent. The Anti-Fade Reagent has anti-oxidant
properties that will retard the oxidative process.
In the preparation of wash buffers, avoid the use of water that may damage Cy5 647. As
noted in the Internet List Serve, MilliQ® water has been shown to damage Cy5
(http://groups.yahoo.com/group/microarray/ messages/2867). Use deionized, reagent
grade water from VWR (Cat. No. RC91505) to prepare wash buffers.
Use a small quantity of dithiothreotol (DTT) to the first two wash buffers used after the
3DNA hybridization. The DTT should be at a final concentration of 0.25 mM. Be sure to
work with fresh DTT, as old or poor quality DTT may cause an increase in background
visible as a “haze” in the Cy3 channel.
Additional Materials:
• cDNA sample
• Reagent Grade Deionized Water (Recommended: VWR Cat No. RC9150-5)
• Glass coverslips (Corning brand)
• 20X SSC (Sodium Citrate/Sodium Chloride) pH 7.0 (175.3 g NaCl, 88.2 g NaCit per Liter)
Cell & Molecular Biology Laboratory
BIOL 406
Hybridization of cDNAS to Yeast Microarrays
• 2X SSC, 0.2% SDS
• 2X SSC buffer
• 0.2X SSC buffer
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buffer
• 100 mM DTT
• 0.1 mg/ml Herring Sperm DNA (blocking agent species specific)
Vortex Mixers
Microarray hybridization chambers
80ºC Heating Blocks
42ºC & 65ºC Water Bath Incubators
Microfuge 4ºC
Microfuges RT
NOTES:
Lab bench and pipettors
Before working with RNA, it is always a good idea to clean the lab bench and pipettors with an
RNase decontamination solution (e.g. Ambion RNase Zap Solution).
Gloves and RNase-free technique
• Wear laboratory gloves at all times during this procedure and change them frequently. They will
protect you from the reagents, and they will protect the RNA from nucleases that are present on
skin.
• Use RNase-free tips to handle the wash solutions and the Elution Solution, and NEVER put
used tips into the kit reagents.
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!
Steps that have already been completed
Prehybridization and Blocking
1. Place slide into a 50 ml tube filled with warm (55ºC) 3x SSC, 0.1% SDS, 0.1 mg/ml sonicated
herring sperm (hs) DNA. The slide must be completely immersed. Two slides can be placed back
to back (ie. arrays facing outward).
2. Agitate gently for 30-60 minutes at room temp.
3. Quickly transfer the slide to a 50 ml tube with dH2O. Dip briefly several times. If you see driedon streaks, you will have streaks in your final scan. If you see streaks, dip several more times in
dH2O
4. Take the slide and place it into an empty 50ml conical tube. Make sure the oligos are “facing
in”. Cap the conical tube and place it into swinging bucket. Spin for 1.5 minutes at 1000rpm.
The idea here is that the force will “push” water droplets down the slide at a constant rate to avoid
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Hybridization of cDNAS to Yeast Microarrays
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streaking. When done, there should be a small volume of water collected at the very bottom of
the tube and the array should be dry.
5. Carefully remove the array (you may need help with tweezers as sometimes the arrays get
slightly stuck) and place into a new 50ml conical tube. Place the tube containing the slide into a
50°C incubator for 10 minutes to equilibrate.
Step 3 cDNA Hybridization
1. Thaw and resuspend the hybridization buffer by heating to 65-70°C for at least 10 minutes or
until completely resuspended. Vortex to ensure that the buffer is resuspended evenly. If
necessary, repeat heating and vortexing until all the material has been resuspended. Microfuge
for 1 minute.
2. For each array, prepare a cDNA Hybridization Mix according to the tables below:
Use Vial 7 Hybridization Buffer
Glass Coverslip Size, mm
Final Hybridization Volume
Concentrated cDNA
LNA dT Blocker (Vial 9)
Nuclease Free Water (Vial 10)
2X Hybridization Buffer (Vial 7)
24x40
34µl
10µl
2µl
5µl
17µl
3. Gently vortex and briefly microfuge the cDNA Hybridization Mix. Incubate the cDNA
Hybridization Mix first at 75-80°C for 10 minutes, and then at the hybridization temperature until
loading the array. Pre-warm the microarrays to the hybridization temperature.
4. Gently vortex and briefly microfuge the cDNA Hybridization Mix. Pipette the cDNA
Hybridization Mix onto one end of the prewarmed microarray (opposite the markings), taking
care to leave behind any precipitate at the bottom of the tube.
5. Place one end of the cover slip onto the end with the sample. Use a fine gauge needle or
single edge razor blade lower the other end of the cover slip but raise it back up after the solution
has made its way about half way, then allow it to settle on the array. This serves to mix the
sample while it is applied to the array.
6. Some chasing of bubbles can be done, but do not make major adjustments to the coverslip
once it is on the array. You want to avoid scratching the array.
7. Place the slide in a hybridization chamber and incubate at 50ºC for 15-16 hours (notes:
different hybridization solutions require different hybe temps; the time of hybridization can be
increased to 2-3 days if care is taken to prevent drying of the array in the chamber).
(Monday’s Lab Starts here)
Wear Powder-Free (Nitrile not latex) gloves when handling arrays!
Always handle arrays and coverslips by their edges.
The oligos are on the side of the slide with the bar code
Cell & Molecular Biology Laboratory
BIOL 406
Hybridization of cDNAS to Yeast Microarrays
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Post cDNA Hybridization Wash
You will need to prepare your wash solutions from lab stock solutions:
20X SSC
10% SDS
100 mM DTT
1. Prewarm 2X SSC, 0.2%SDS (~40 ml x 2) wash buffer to 50°C.
2. Remove the coverslip by washing the array in a 50 ml tube with the prewarmed 2X SSC, 0.2%
SDS for 2-5 minutes or until the coverslip floats off.
3. Wash for 15 minutes in prewarmed 2X SSC, 0.2%SDS (~40 ml).
4. Wash for 15 minutes in 2X SSC at room temperature (~40 ml).
5. Wash for 15 minutes in 0.2X SSC at room temperature (~40 ml).
6. Transfer the array to a dry 50mL centrifuge tube, orienting the slide so that any label is at the
bottom of the tube. Immediately centrifuge without the tube cap for 2 minutes at 800-1000 RPM to
dry the slide (any delay in this step may result in high background). Avoid contact with the array
surface. Agitation during washing may also help to reduce background due to non-specific
binding to the surface of the array.
Practice applying coverslips to regular slides using 40ul of 0.1% SDS to
mimic the microarray solutions.
Proceed to Step 4: 3DNA Hybridization and Wash
Step 4: 3DNA Hybridization and Wash
Caution: In order to protect the Cy dyes, keep the hybridized slides in the dark as
much as possible. Use of aluminum foil sleeves on 50 ml tubes is recommended.
We will reduce light as much as possible in the lab to avoid degrading the fluors.
1. Prepare the 3DNA Array 350 Capture Reagent (Vial 1). It is necessary to break up aggregates
that may form as a result of the freezing process.
a. Thaw the 3DNA Array 350 Capture Reagent (Vial 1) in the dark at room temperature
for 20 minutes.
b. Vortex at the maximum setting for 3 seconds and microfuge briefly.
c. Incubate at 50-55°C for 10 minutes.
d. Vortex at the maximum setting for 3-5 seconds.
e. Microfuge the tube briefly to collect the contents at the bottom.
Be sure to check the sample for aggregates prior to use and repeat vortex mixing if necessary.
Aggregates may appear as small air bubbles or flakes at the side of the tube below the surface of
the solution. Repeat steps a-e if necessary.
2. Thaw and resuspend the 2X Hybridization Buffer (Vial 7) by heating to 70°C for at least 10
minutes or until completely resuspended. Vortex to ensure that the components are resuspended
evenly. If necessary, repeat heating and vortexing until all the material has been resuspended.
Microfuge for 1 minute.
3. Prepare a stock solution of Anti-Fade Reagent (Vial 8) by combining 1µl of Anti-Fade with
100µl of the 2X Hybridization Buffer (Vial 7). The Anti-Fade Reagent helps reduce fading of the
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Hybridization of cDNAS to Yeast Microarrays
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fluorescent dyes both during and after the hybridization process. Store any unused hybridization
buffer containing Anti-Fade Reagent at –20ºC. (Only one group needs to do this as you only need
17 µl per slide.)
4. For each array, prepare a 3DNA Hybridization Mix according to the table below.
Glass Coverslip Size, mm
Final Hybridization Volume
3DNA Capture Reagent #1 (Vial 1)
3DNA Capture Reagent #2 (Vial 1)
Nuclease Free Water (Vial 10)
2X Hybridization Buffer (7) with Anti-Fade Reagent from step 3
24x40
34µl
2.5µl
2.5µl
12µl
17µl
5. Gently vortex and briefly microfuge the 3DNA Hybridization Mix. Incubate the 3DNA
Hybridization Mix first at 80°C for 10 minutes, and then at the hybridization temperature until
loading the array. Pre-warm the microarrays to the hybridization temperature (50ºC).
6. Gently vortex and briefly microfuge the 3DNA Hybridization Mix. Place the microarray in the
bottom half of the hybridization chamber. Add the 3DNA Hybridization Mix to a prewarmed
microarray, taking care to leave behind any precipitate at the bottom of the tube.
7. Apply a glass coverslip to the array the same way you practiced. Place one end of the cover
slip onto the end with the sample. Use a fine gauge needle or single edge razor blade lower the
other end of the cover slip but raise it back up after the solution has made its way about half way,
then allow it to settle on the array. This serves to mix the sample while it is applied to the array.
Seal the hybridization chamber and incubate the array for 3-4 hours in a dark humidified chamber
at 50ºC.
NOTE:
Subsequent steps will be carried out for you,
so that slides can be shipped for reading.
Post 3DNA Hybridization Wash
Additional protection of the fluors is afforded by the 1 mM DTT added to each
wash solution. It is very important that the microarray not be allowed to dry
between washes.
Perform the following steps in the dark to avoid degradation and fading of the fluorescent
dyes
1. Prewarm the 2X SSC, 0.2%SDS, 0.5mM DTT (~40 ml x 2) wash buffer at 65°C
2. Remove the coverslip by washing the array in prewarmed 2X SSC, 0.2% SDS, 0.5 mM DTT
(~40 ml) for 2-5 minutes or until the coverslip floats off.*
3. Wash for 15 minutes in prewarmed 2X SSC, 0.2%SDS, 0.5 mM DTT (~40 ml). (DTT is
included to help prevent the fading of the Cy5 fluorescent signal.)
4. Wash for 10-15 minutes in 2X SSC, 0.5 mM DTT (~40 ml) at room temperature.
5. Wash for 10-15 minutes in 0.2X SSC (~40 ml) at room temperature.
6. Transfer the array to a dry 50mL centrifuge tube, orienting the slide so that any label is at the
bottom of the tube. Immediately centrifuge without the tube cap for 2 minutes at 800-1000 RPM to
dry the slide (any delay in this step may result in high background). Avoid contact with the array
Cell & Molecular Biology Laboratory
BIOL 406
Hybridization of cDNAS to Yeast Microarrays
surface. Agitation during washing may also help to reduce background due to non-specific
binding to the surface of the array. Proceed to Signal Detection.
SIGNAL DETECTION
IMPORTANT: Store the array in the dark until scanned. The fluorescence of the 3DNA
reagents, especially Cy5 can diminish rapidly even in ambient light because of oxidation.
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