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Hybridization of cDNAS to Yeast Microarrays - Laboratory | BIOL 406, Lab Reports of Cell Biology

Ramapo College of New Jersey (RCNJ)Cell Biology

Material Type: Lab; Class: CELL AND MOLECULAR BIOLOGY: LECTURE AND LAB; Subject: Biology; University: Ramapo College of New Jersey; Term: Unknown 1989;

Typology: Lab Reports

Pre 2010

Uploaded on 08/18/2009

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Cell & Molecular Biology Laboratory
BIOL 406 Hybridization of cDNAS to Yeast Microarrays
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.
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BIOL 406 Hybridization of cDNAS to Yeast Microarrays

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 ( 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.

BIOL 406 Hybridization of cDNAS to Yeast Microarrays

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

BIOL 406 Hybridization of cDNAS to Yeast Microarrays

  • 2X SSC, 0.2% SDS buffer
  • 2X SSC buffer
  • 0.2X SSC 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 dH 2 O. Dip briefly several times. If you see dried- on streaks, you will have streaks in your final scan. If you see streaks, dip several more times in dH 2 O
  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

BIOL 406 Hybridization of cDNAS to Yeast Microarrays

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.

  1. 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 24x Final Hybridization Volume 34 μ l Concentrated cDNA 10μl LNA dT Blocker (Vial 9) 2μl Nuclease Free Water (Vial 10) 5μl 2X Hybridization Buffer (Vial 7) 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

BIOL 406 Hybridization of cDNAS to Yeast Microarrays

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.)

  1. For each array, prepare a 3DNA Hybridization Mix according to the table below. Glass Coverslip Size, mm 24x Final Hybridization Volume 34 μ l 3DNA Capture Reagent #1 (Vial 1) 2.5μl 3DNA Capture Reagent #2 (Vial 1) 2.5μl Nuclease Free Water (Vial 10) 12μl 2X Hybridization Buffer (7) with Anti-Fade Reagent from step 3 17μl
  2. 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).
  3. 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.
  4. 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

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.