Microarray analysis of oxygen rhythms in yeast, 2002.  

Laboratory 1: Discussion of concepts and techniques.

Experimental Samples: our class is collaborating with Dr. Robert Klevecz from City of Hope to analyze rhythmic gene expression in continuously cultured yeast cells (see references below).  This will be original research in collaboration with a noted cancer molecular/cellular biology lab currently studying yeast.

Discussion: students from class will be asked to discuss questions that might arise about rhythmic biological processes and methods by which one might examine these questions.  Students will also be asked to discuss the nature of microarray analysis, its strengths and its weaknesses.  Each student should write up these discussions in his/her laboratory notebook.  Readings for first lab discussion: E-res readings 2 and 3 for class (Gerhold on microarray background and White on application of microarray analysis to development of flies); ms. from Douglas and Klevecz (handout in lab).  Additional materials to be used in passing in the first lab and discussed more later: E-res paper 34 on gene regulation's role in rhythmic processes, and De Risi, J, Iyer, V and Brown, P, Science 278:680-686 (1997) on diauxie in yeast studied via microarrays (available on line, use PubMed from the library's web site, or you can go to the SMD site, choose "literature" and browse the list to find this paper).

Microarray Analysis, laboratories 2,3: Total RNA Preparation Using Hot Phenol and Quality Control of RNA

 Laboratory period 2, Hot Phenol Extraction of Yeast to Prepare Total RNA.  Use gloves, clean area, sterile, RNase-free equipment; these precautions are known as RNA paranoia!!  

 First, before beginning the lab work, set up RNA PARANOIA and obtain samples:

1. Clean your lab bench with RnaseErase and cover it with a sterile pad 
2. Put on gloves; don't scratch your nose or touch anything like paper (contains RNase!) after you are gloved.
3. Obtain tubes of AE buffer, SDS, and phenol  at room temperature.
4. You will be using two frozen pellets from two samples of cells obtained from a chemostat culture of yeast that maintains the culture at 1x10⁹ cells per ml.  One of these will be at the peak of the cycle or at the trough (bottom) of the cycle.  The other will be at an intermediate point in the cycle.  These samples will be provided in a dry ice bucket in the laboratory. 

Procedure

1.      Scale: 0.5 ml of the chemostat culture, amounting to 5 x 10⁸ cells; this compares with starting with cells frozen from 30 ml log phase culture with an absorbance at 600 nm of 0.7.  ( A general fudge factor, that should be checked for each yeast strain used, is that 1 ml of cells at absorbance at 600 nm of 0.7 is 1 x 10⁷ cells, approximately).  If the pellets provided are not in 15 ml disposable centrifuge tubes, then transfer each to a clean 15 ml tube.

2.      Add to each pellet of frozen cells using sterile disposable pipets and Pi pump, 3 ml AE buffer; vortex to thaw completely.  Do not put on ice, keep at room temperature.

3.      Add 0.5 ml 10% SDS (again, do not put on ice).

4.      Add 3.0 ml acidic phenol in the hood  using 3 round of transfer with 100 ul from the P1000 pipettor (take the BOTTOM layer; phenol has been pre-equilibrated with MQ water and acidified); mix; you will see the protein of the cells become a white precipitate. 

5.      Hot phenol extraction: place at 65°C in TempBlok for 30 min, invert to mix every 5 min.  This step will thoroughly denature the proteins and help them to separate from any nucleic acids with which they are coprecipitated.  

6.      Cool down: 5 min on ice.

7.      Spin 10 min at 2500 RPM in clinical centrifuge; share the centrifuge.

8.      Carefully pipet up the supernatant (upper layer) using a P1000 without dipping into or transferring any of the white interface layer.  Transfer the supernatant to a new 15 ml tube.

9.      Chloroform extraction: Add equal volume (3 ml) of chloroform to tube of supernatant (in the hood again), invert several times to mix.

10.  Spin 10 min ( be sure to balance two of your tubes and balance your third with another group’s tube) in clinical centrifuge.

11.  While spinning, add 1/10 vol of 3M Sodium Acetate (NaOAc) (300ul) to a new 15 ml tube.  Also, get a tube of 100% ethanol and place in your ice bucket to chill for step 16.

12.  Transfer upper layer from the chloroform extraction into tube with NaOAc prepared  in step 11.

13.  Add 3.3 ml of room temperature isopropanol to top of tube, mix, precipitate in freezer (-20°C 30 minutes or O/N).  From this point on in the procedure, keep in an ice bucket between steps.

14.  Spin 30 min at 3K in clinical centrifuge (if possible at 4°C; ask instructor if we have a clinical centrifuge in Dr. Becker's cold room).

15.  With a quick motion, dump out the liquid. 

16.  Resuspend the pellet in 250 ul of cold 100% ethanol.

17.  Transfer resuspended pellet to a sterile RNase free 1.5 ml tube.

18.  Add 250 ul cold 100% ethanol to the original tube, then pipet out and add to the tube from step 17.  If you still have white chunky material in the original tube, repeat this step to get it all into the microfuge tube.  Store at –20 in freezer until next period.

Second laboratory period.  Collecting and redissolving RNA; quality control.  Use RNA paranoia!!  We will start with a brief discussion of what we did last week and where we are in procedure; see lab schedule for details!

19.  Spin down the pellets  in a 4°C microfuge.

20.  Dry pellet in SpeedVac, temp setting at 30°C, in Martinez Lab (try to organize your selves so that only one of us is hovering in the DM lab at once; they probably would like to do research there!).  Stop immediately when dry. (Look at in 15 minutes; if you have a lot it may take 1 hour).

21.  Resuspend in 150 ml DEPC water.  Allow to sit at room temperature for 1 hr – (or quicker, possibly 15 minutes, use frequent resuspension with pipetman).  Make sure dissolved!  You may have clear lumps but these should become completely dissolved with more time.

22.  Quantitate on spectrophotometer.  RNA is quantitated by its absorbance at 260 nm, A_260. (Expected yield ~ 0.9 mg)

    a. Take 2 ul of each sample and add 998 ul of MilliQ water (same as diluting 1:500 in MQ water).  Keep the rest of your sample on ice until you are sure you don't need more for reading absorbance and in addition, the gel sample has been removed from the RNA tube (see under 23 below).

    b. Mix the 1:500 dilution thoroughly and read the absorbance at 260 nm. against a blank of water. Do not discard your sample, since you may have to add more RNA and reread it. Show your results to the instructor, who will tell you how much more to add from your RNA stock if you need to reread the absorbance.

    c. Calculate the expected absorbance. If the original solution has 1.5 mg in 0.5 ml (concentration of 3 mg/ml, yield of 1.5 mg), and you have diluted 1:500, here is how you calculate the expected absorbance: (3 mg/ml)/500 dilution factor = 0.006 mg/ml.   Now multiply by the absorbance of a known solution: 25 A₂₆₀ at 1 mg/ml.  After multiplication, you get 0.150 as the predicted A₂₆₀. Therefore if your yield is much lower than this, say only 50 ug rather than 1500 mg, you will need to add more RNA (about 20 to30 times more) to the absorbance dilution to see an absorbance over 0.100.

    d. Calculate the concentration of RNA in your sample; save the dilution in case you may have to add more RNA.  Once you have observed the absorbance of the 1:500 dilution at 260 nm, consult the instructor to see if you need to repeat the measurement with more sample added.  Next, divide by 25 to correct to  mg of RNA, and multiply by the dilution factor 500* (actually, you can cancel out these two and just multiply by 20) to get the mg/ml.  Since there are 1000 ug per mg, you can multiply this by 1000 to get the ug/ml.   Post your results (concentration in ug/ml, total ug obtained, which is the concentration times the volume in ml) on the board in front of the lab room.  

            *If you have added more RNA, you need to adjust the value of this to 100, 250 or whatever is appropriate for the actual dilution you used.

    e.  Calculate, using your ug/ml concentration from part d, the volume (number of ul) you would need to obtain 20ug of total RNA for the next laboratory.  Record this volume in your notebook and consult the instructor to see if you need to make your RNA more concentrated.  

   23.   Run a gel to find out if the RNA is degraded.

    a. A 1.5% agarose gel has been prepared that contains ethidium bromide. As you recall from Bio40, this dye is a carcinogen, so be especially careful not to expose yourself to it (RNA paranoia is good enough but changes gloves after interacting with the gel/buffer; just don't dip your gloved hand into the tank buffer and then keep on the same gloves, spreading the dye around the lab.)

    b. Take 5 ul of the undiluted RNA sample and add 1 ul of 6x tracking dye. If you had low absorbance, take your calculations of the volume for 20 ug to the instructor and see if you have enough to increase the amount of RNA loaded on the gel.  If you did not need to concentrate your RNA, you should take it back to the -20 degree freezer now, using a gloved hand.  If you did need to concentrate it, then you should refreeze it as soon as possible after concentrating on the Speed Vac (discuss plans for concentrating with the instructor).

    c. Place your sample into a well of the gel and label on the diagram of the gel where it is placed. 

    d. One group should add 4 ul of the RNA standard to an empty slot. 

    e. When all groups' samples are loaded, put the lid on the gel apparatus and run it for 1 hour at 80 V. During the hour, we will meet in the Thille 103 classroom to continue our discussion of background for rhythmic biological processes.

    f.  Photograph the gel on the UV lamp (make a photo for each student's notebook and one for the instructor). If one or more of your RNAs looks degraded (see sample gel drawn below), consult the instructor about whether or not to use this sample next week for the cDNA synthesis.  Or, we can take the gel to SS basement and look at it on LH's imager.

24. Pool part of each sample for the control mixed sample; this control will be used on each of your microarrays.  The pool is prepared as a class.  LH will set up, in the ice bucket, a clean sterile microfuge tube labelled "control mixture".  After you have completed step 23 f above, you will know which of your RNAs are "good" samples.  LH may or may not have you add all of your good samples to the mixture; the mixture needs to represent the average of all time points we will use, so it may be that one or more will be omitted.  Therefore, look at the list provided next to the ice bucket to see which of your samples will be used.  With gloved hands, obtain those samples from the -20 freezer into an ice bucket and let them thaw.  Next to your sample on that list, fill in the number of ul that correspond to 20 ug of total RNA, and add that number of ul to the tube (make sure you have enough for a 20 ug individual sample left!  You may need to add half of your sample; if problems arise, please consult the instructor).  Please measure very carefully and dispense all of the ul needed for the 20 ug amount of sample to the mixture tube.  Check off your samples from the list beside the ice bucket when added.  The last person to add a sample, please tell the instructor so she can Speed Vac the sample down to a smaller volume for use next week.

Lab notes: Put your ug/ml of RNA on the board in lab; copy down all of these numbers into your notebook.  Look at the yields and look at the samples on the gel photograph.  Note the quality of each RNA sample in your notebook and give your reasons.  Note which of your samples you will be using to continue the experiment.

Class data for 2002: The samples contain two rRNA bands and some bands larger than those.  There is an mRNA haze behind the rRNA bands, meaning that large mRNAs were obtained.  There is some DNA on the slots of these 1.5% gels in some of the samples.  In an ideal gel, there would be a stronger stain for the larger of the two rRNAs indicating no losses of this large molecule.  In these gels, few samples meet this stringent criterion. Our collaborators at City of Hope say that hot phenol may usually cause this effect.  Yields of these samples are indicated below each position.

      std       1      4       7       8      9     10                                  std             3       6        2       5        

yield, ug:     114  189   393  204   126  204                                              86      128   96     108              

Materials (preparative notes)

Period 1:

RNase Away

Kim Wipes

Bench cote rectangles (Bring over 20 for first lab; extras will be kept in cabinets between labs)

Dry ice bucket of samples of frozen yeast cells, 3 per person

Fume hood

Micropipettors (from advanced lab box; P20, P200, P1000 per person)

TempBlok set to 65 degrees

Filter tips (sterile, P20, P200,  and P1000 sizes); need 6 boxes each

Falcon tubes (15 ml centrifuge tubes): 2 bags of 40

Eppendorf tubes (beakers full of multicolored 1.5 ml tubes and topped with two layers of foil, then autoclaved; bags of tubes are in Bio 164 cabinet.

Ice buckets (top of Thille 113 cabinets), 6 (1 per students and 1 for reagents)

Freezer boxes, 6 (OK to reuse old ones)

Clinical centrifuge (ideally, at least one at room temp and one in cold room)

Acidic phenol (put in hood; screw cap bottle with an alliquot from lab supply, don't put it all out)

10% SDS (tubes of 1.5 ml, make 7)

DEPC Water (Milli Q water required) (Tubes of 7.5 ml, make 7)

3M NaOAc (sodium acetate) (tubes of 1ml, make 7)

Isopropanol (Sigma I9516) (put bottle in hood with clean beaker)

Ethanol (Pharmco, USP ethyl alcohol 200 proof) (tubes of 1.5 ml, make 7) 

Chloroform (Sigma C2432) (put bottle in hood with clean beaker)

AE buffer (recipe follows) (tubes of 7.5 ml, make 7)

Preparation of reagents needed:

AE Buffer Prep (AE= 50 mM sodium acetate (NaOAc) pH 5.2,  10mM EDTA)

    Per 500 ml

    8.33 ml NaOAc 3M

    10 ml EDTA 0.5 M pH 8 (prepared according to Maniatis)

acidic phenol: saturate phenol with pH 6 MilliQ water (check with MW first)    

DEPC water: (Recipe for1 L, you can actually make 300 ml stock.  Keep in Rm 5 and dispense as called for to the class.

    1ml diethyl pyrocarbonate (Sigma D5758)

    1L MilliQ Water

    Stir overnight in the fume hood, autoclave to inactivate DEPC.

Loading dye for next week: see recipe below; make in first week and filter a couple of days later so completely                 dissolved.

  Second laboratory, MATERIALS:

RNase Away

Bench coat (extra from last time)

Speed Vac in Dr. Martinez' lab; check if OK to use Weds PM.

Pipettors: P2, P20, P200, P1000 (5 sets)

Refrigerated microfuge (set the refrig microfuge to 4 degrees and run it to chill down)

Check if adequate sterile 1.5 ml tubes (probably enough).

Check if adequate filter tips (probably enough; also need P2 tips this time, 5 boxes if possible or they can share).

Disposable UV cuvettes

Squirt bottle of deionized water to use at spectrophotometer.

Kim Wipes to use at spectrophotometer.

DEPC water, 1 ml, 7 tubes (OK to use from last week's stock)

Loading dye for RNA: make Maniatis recipe dye with glycerol, bromphenol blue, and xylene cyanole; use DEPC water, filter sterilize after completely dissolved.

RNA standard (frozen; put in course freezer box in the -20 freezer between the Thille labs on the Bio164 shelf)

1.5 % agarose gel, 0.5 x TBE, with ethidium bromide in gel, in tank with tank buffer in back of lab (near blackboard).

Blank paper labeled : Gel loading diagram, next to gel box

UV spectrophotometer...check availability of the one from Rm 18 during Weds PM.

Microarray Reverse Transcriptase cDNA Probe Synthesis

Laboratory 4    ***Use RNA paranoia, gloves, bench coat, no fingerprints, etc***

 1.Anneal Primers (30 minutes, 1:20-1:50)

a. Set up ice bucket; using gloved hand, get each of your good RNA samples (up to two) from –20 freezer and place on ice.  The pool sample has been aliquotted into samples with 20 ug each; take as many of these as you have good samples. Your sample number could therefore range from 2 (one good sample and one pool) to 4 (three good samples and three pools).  The amount of liquid (in ul) of the pool sample after Speed Vac  that corresponds to 20 ug of total RNA mix will be posted on the board in the lab.

b. Bring volume containing 20 ug of total RNA  to 14.5ml using DEPC water.  You will use each of the two samples you prepared that passed quality control tests, and for each sample, you will prepare a mixed total RNA partner.

c. Add 1ml oligo dT primers to each tube (from ice bucket in front of lab; share 1 tube).

d. Mix, quick spin

e. Incubate at 70 °C for 10 min.

f. Chill on Ice 10 min.

cDNA Synthesis (2 H, 25 minutes, 1:50-4:15)

2. To each reaction add 14.5 ml of the following mix:

                                                            14.5                 4(14.5 ml aliquots)

   a. Mix

   b. Quick spin, then incubate at 42 °C for 2 hours.

*****During the 2 hour period, we will go to the Thille 103 classroom and discuss some background for the rhythmic cycling experiment.

3. Hydrolyze RNA by adding: 1 ml 1M NaOH and 3.2 ml 0.5 M EDTA pH 8 to each sample

    a. Mix, quick spin, and incubate 15 min at 65°C ; after this step, you should have cDNA without any RNA.

    b. Neutralize:  add 25 ml 1M HEPES, mix, quick spin.

4A.  Reaction cleanup using Zymo column (Note: directions differ from those included with columns!! Use these!!).

   a. Add 1 ml of Zymo  Binding Buffer to each tube, mixing well.

   b.  Load 500 ul of your sample onto the Zymo Clean and Concentrator-5 column and spin at 6000 rpm for 30 seconds.

   c.  Add the remaining liquid from your sample from step 1 onto the top of the Zymo column and spin at 6000 rpm for 30 seconds again.  No need to change the collecting tube since it holds more than 1 ml.

   d.  Discard the ~ 1 ml of flow through; your cDNA is bound to the column.

   e.  Wash bound materials by adding to the top of the column  600 ul of Wash Buffer (containing Ethanol).  Spin wash buffer through column at 14,000 rpm (or top speed in microfuge) for 60 seconds.

   f.  Remove flow through liquid and spin the column again to remove any residual solution on the column itself.

   g.  Remove all liquid from the bottom tube, or change bottom tubes if an extra catch tube is available.

   h.  Elute the cDNA from the column by adding 8 ul of Milli Q (MQ) water to the top of the column and letting sit at room temperature for 1 minute.  This is important!  

  i. Spin the first part of the elution buffer through after the 1 minute period, at top speed for 30 seconds.

  j.  Elute again with 6 ul of MQ water, to get a more quantitative yield.  Let sit for 1 minute. Spin at top speed for 60 seconds. 

  k.  Store your 14 ul of eluted cDNA in a clearly labeled tube with your initials, group, date, and sample number, at -20 degrees in the freezer until the next laboratory period.

4B.  SKIP THIS STEP IF YOU DID ZYMO COLUMN! Alternate Cleanup using Microcon-30 Spin Dialysis ( 40 

minutes, 4:15-4:55) SKIP  SKIP   SKIP SKIP

   a. NO..don't use these steps if you have used the Zymo column!  This is an alternative method.  Fill one Microcon-30 concentrator per sample with 450 ml MQ water

   b. Add neutralized reaction.

   c.  Spin at 10,000 g (not rpms!) in Eppendorf microfuge for 9 min; share microfuge!  

   d.  Dump flow-through that has collected in the bottom tube.  You should see only a small amount of liquid (e.g. 5ml of liquid) on the membrane at the top, but it should not be dry!.  This step is concentrating the cDNA, not binding it to the membrane.

     e. Repeat process 2X, refilling original filter with 500ml MQ water and spinning 8 min.  (If some tubes have less volume than others, add a few more drops of water before subsequent spins).  

   f. After final spin, add 50ml of MQ water.

   g. Invert filter into collection tube and spin for 1 min.

   h. Dry  the 50 ul of eluate you obtained from the top of the filter for 5 minutes in speed-vac in Dr. Martinez’ laboratory.  

  i.  Store pellets at –20 °C.

  Materials:  

Check bench coat supply, tip supply, sterile tube supply.

Micropipettors

Temp blok set at 65 degrees

Microfuge (green one that belongs to Bi164)

Ice buckets from top shelves in th113

DEPC water (7 tubes of 1.5 ml, freshly prepared not reused from last time)

Milli Q water (7 tubes of 1.5 ml, freshly prepared)

OligodT primer (20 mers) ; one tube in ice bucket (MY: make a box of things that I should move to an ice bucket for lab)

Stratascript II Rtase (Stratagene 600085); one tube in ice bucket, MY put in freezer box with OligodT

dNTP set ultrapure (Amersham)(one set in same box in freezer with above items) 

Qiaquick PCR purification kit (Quiagen 28106)

5MG 5-(3-aminoallyl)-2'-deoxyuridine 5' triphosphate sodium salt (Sigma A0410) one solution in same freezer box

Microcon 30 (Amicon 42410) or Zymo columns sufficient for all samples.

 Preparation of reagents:

How to make oligo dT/N6:

Order  dT oligo, purified 20 mers, from Invitrogen.

Resuspend the oligo in MQ water to a final concentration of 10 mg/ml.

Resuspend pd(N)6 random hexamer in DEPC water to a final concentration of 10 mg/ml.

Combine the oligo stock with equal volume of pd(N)6 random hexamer to make a final concentration of 5mg/ml each.

  NOTE we are not using the N6 primers, just the OligodT

A ratio of 2 aa-dUTP’s:3 dTTP’s was optimized for our yeast chips.  Altering the ratio to 3:2, 1:4 or 4:1 may help increase signal.

Coupling of activated fluorescent dyes to cDNA and hybridization of microarrays

(laboratory 5 plus washing the morning after laboratory; repeated with modifications underlined in laboratory 6)

You have DNA not RNA, but still use normal molecular biology good technique ( use clean sterile tips, wear gloves).

Cy Dye Coupling

1. You will be labeling each of your high quality RNA samples with one of the two dyes (Cy3, green or Cy5, red).  In addition, you will be labeling a matching mixture sample for each test RNA.   A table showing what dye to use for each sample will be discussed at the start of the laboratory. You need to make the test RNA (your sample) and the control RNA (mixture or log phase RNA) different colors so they can be told apart on the hybridized array.  See posted on the board directions for which color to use for each of your test RNAs; then make the control for it the other color.  
2. Take one tube of the correct Cy dye for each of your samples and each of your mixed controls.  Allow tubes come to room temp in your rack covered with foil. [The dye is dry; you will resuspend it later.  Label each dye tube on both top and side with the sample that will be coupled to its dye and with your group.] Dye will be resuspended in 100 mM sodium bicarbonate pH 9.0 when you receive it.  Keep it at room temperature until used, under foil.
3. Directions if you used a Zymo column last week: Your samples were reverse transcribed by Michelle Wu.  Your cDNA, synthesized with a reactive nucleotide that can bind to the activated dyes today, was eluted from the Zymo column in 8 ul + 6 ul, for a total of 14 ul.  To adjust the concentration of this sample to 0.1 M [0.05] M Sodium Bicarbonate, pH 9.0, add to this sample [0.7] about 1.3 ul, check with MW for exact number of 1 M sodium bicarbonate pH 9.0 using the P2 pipettor.  Mix gently by finger vortexing, no need to place on nutator.  [( Directions if you used a MicroCon column last week (omit in 2002): Resuspend each of your cDNA pellets (that you dried during the last laboratory and stored at -20 degrees C) in 9 ml 0.05M NaBicarbonate Buffer, pH 9.0.  Use nutator to mix and let tubes rotate for 10-15’ at room temperature to ensure resuspension.)]
4. Dye is resuspended skip resuspension.  Add 2ul of the dye to the correct sample and mix.[Meanwhile, resuspend each monofunctional NHS-ester Cy3 or Cy5 dye in 1.25 ml DMSO; keep under foil until you complete the resuspension time for the cDNA, then simply transfer your cDNA + Bicarbonate Buffer into the aliquot of dye, making sure to match the sample transferred to the tube’s label.] 
5. Let your samples incubate 1 hour at RT in dark (under foil, make sure of no light leaks).

 Hybridization Chamber Preparation

In the procedure to follow, you are trying to the maximum of your ability to keep dust off your array slide and coverslip.  Let it be open to the air as little time as possible!

1. Carefully lay out your chamber.  Next to it on the blue mat lay out the microarray slide.

2. Cover the array slide with the chamber lid  to protect the array before proceeding to clean the cover slips.

3. Obtain two lifter coverslips for use with your microarray, using gloved hands.

4. Clean slips with 95% ethanol designated for microarray experiments (Mallinckrodt Reagent Alcohol, MK70194).

5. Dry gently with kimwipes, and a few blasts of whoosh duster if needed. Never hold the Whoosh duster too close to the slip or tilt the can while spraying.

6. Now you are ready to apply the cover slips to the array.  Uncover the array slide, placing the chamber cover on top of the chamber but not clampling them together.  

7. Hold the cover slip by its sides and lay one edge down on the correct place on the array slide.  Supporting that edge with your gloved index finger, gently let go of the cover slip to cover the array on one end.  Repeat for the other end of the slide with the second cover slip.

8. Allow to stand undisturbed unti the probe is ready for hybridization

Cleaning up the Coupled cDNAs

Before combining Cy3 and Cy5 samples for hybridizations the reactions must be cleaned up to prevent cross coupling. To remove unincorporated/quenched Cy dyes proceed with the Zymo column [with Qia-Quick PCR Purification Kit (QIAGEN) as follows:]

1. To each dye/cDNA mixture after incubation, add 1 ml DNA binding buffer [70ml MQ water].
2. [Add 500ml Buffer PB.] Skip this step
3. Apply to Zymo column  530 ul of sample (about 1/2 of volume),[Qia-Quick column] and spin in microfuge at 6K rpm[13K rpm] for 30-60 sec.
4. [Aspirate off flow-thru.]
5. Apply the rest of the sample to the column [Add 750 ml Buffer PE] and spin at 6K rpm for 30-60 sec.
6. Aspirate off flow-thru [and repeat]. Wash each column with 600 ul of wash buffer; spin max speed for 30-60 seconds.
7. Aspirate flow-thru and spin for additional 60 sec at high speed to dry column.
8. Transfer column to fresh 1.5ml tube.
9. [Add 30ml Buffer EB to center of filter and let sit 60 sec at RT.] Arrange the columns in pairs that will be hybridized on the same array.  Add 6 ul MQ water to the column for the Cy3 labeled sample in each of the pairs.  DO NOT elute the Cy5 samples yet!!  Incubate at room temperature for 1 minute to allow the labeled probe to dissolve off the filter.
10. Spin at 13K for 60 sec.
11. Take the eluted Cy3 sample from each pair and apply to the partner Cy5 column.  Incubate for 1 minute and spin top speed 30-60 seconds as before. [Repeat elution step again with another 30 ml of Buffer EB].
12. Transfer all Cy5 columns to fresh 1.5 ml microfuge tubes, labeling them to make sure that they stay paired correctly.  Elute these Cy5 columns with 5 ul of MQ water.  Incubate 1 minute and spin at top speed 60 seconds.[Combine each pair for one array of Cy3 and Cy5 samples together after cleaning (total of 120 ml)]
13. Take the eluted Cy5 columns you just prepared and apply to its paired Cy3 column.  Move the column to the top of the original 1.5 ml tube from steps 9-11 (with the Cy3 first, then the Cy5 elution from the same pair of samples). Let sit 1 minute, spin 60 seconds at top speed.  You should now have about 7.5 ul of sample with both labeled probes for one array 

 Hybridization Preparation

 The following procedure works well with ISB chips printed with 70-mer oligonucleotides,  The EasyHyb disrupts the water structure more, so the hybridization temperature will be lower in this case.  Copy down the directions from the board if we use this method, instead of following the method below in steps 1-3; instead 'rejoin' this protocol in step 4.

1. Skip this step.[Concentrate the probe (combined Cy3 and Cy5) in a Microcon-30.  Spin at 10,000g for 5 min (or when the volume has come down to about 5 ml, but NOT dried onto the filter!)  Show to LH, who will recommend further 30 second bouts of centrifugation until the sample looks perfectly concentrated.  It will resemble a liquid bubble on a small part of one side of the filter; check with LH to have her verify that it is concentrated enough so it won't dilute the hyb buffer and make the hyb ineffective.]  
2. Add to the [few] ~7.5  ul left 1.5 ul of 10 mg/ml PolyA.  This will hybridize with oligo dT and  reduce the non-mRNA-specific hybridization, so we can see the specific hybridization more easily.  Measure and record the volume of (polyA plus cDNA) while you are transferring it to a 0.5 ml microfuge/PCR tube.   Check with MW and LH to see if we will be trying 2x Dig Easy hyb buffer today.  If so, you will take 15 ul of 2x Dig Easy hyb buffer into your sample, and make the whole sample up to 30 ul with MQ water.  [lSubtract the volume you transferred  from 40 ul to prepare for the next step.] 
3. [Add enough DIG Easy Hyb to bring the mixture up to 40 ul, enough to go under one of our lifter coverslips comfortably.] We will try 30 ul of hyb this time, assuming we are using the 2x hyb buffer. 
4. Incubate the probe mixture at 100ºC for 2 minutes in the PCR machine (program name: BOIL) to separate the strands.
5. Spin down the probe and allow to cool.  
6. Add 12 ml MQ water to each of the small round wells in the hybridization chamber to help the slide stay hydrated and not have the hybridization mixture pull away from the edges of the array.
7. Make a note of which end of the slide (refer to the numbered end of the slide as the "bottom" of it) will receive each sample, and follow that note carefully in this step.  Apply the first sample pair probe to one end of the prepared microarray by sliding the lifter cover slip slightly to the side and placing the tip you are using for the transfer onto the slight edge of exposed area on the slide itself next to where the array is printed.  
8. Apply  probe to the other end of the prepared microarray.  Recheck the tubes to make sure you have loaded correctly; if not, change your written description of how you loaded the slide. If you get mixed up here, it will be impossible to interpret the data, so be careful about your written notes! 
9. After both ends are loaded with probe, immediately seal the chamber and incubate at 37 degrees if using ISB array with preparation as given on the board, or (in 63ºC water bath if using Stanford array with a different hyb mix recipe)  Record the time you began incubation.  
10. Hybridize for 16 hrs (e.g. 5 PM to 9AM).

    WASHING THE MICROARRAY AFTER HYBRIDIZATION

The portion of the cy dye-labeled cDNA that did not hybridize with the oligos printed on the slide need to be washed off before we can scan the slide.  All of the stock solutions for the wash solutions below should be filtered before use.

1. Prepare glass slide dishes by rinsing with distilled water followed by MQ water (It pays to have clean glassware!)

2. Prepare wash solutions in the round glass dishes, with each dish having its own rack.  Use the designated glass cylinder to measure out the MQ water for the solutions.

    a. Wash solution I: 680 ml MQ water                    b.  Wash solution II:  700 ml MQ water

                                    20 ml 20x SSC                                                          2 ml 20xSSC

                                      1 ml 10% SDS

3.  Carefully remove an array from the waterbath, keeping the chamber level.  Dry completely with paper towels and wick away any water from chamber seams.

4. Unclamp chamber and remove array.  Have a pair of forceps handy; if water has pooled under the slide, these will help to pry the array away from the chamber.

5.  Quickly remove another array from its chamber.  It is recommended to wash no more than two arrays at once to decrease the time the first array sits out after opening the chamber.  On the other hand, MW recommends to open all the chambers and get the arrays ready, rather than submerging the first array and having it wait too long in Wash Solution 1.  

6.  Keep arrays level when submerging in Wash Solution I.  Once submerged, tilt array and gently dump off the cover slip.  It may be necessary to swish array under solution to dislodge the slip.

7.  When all chips are in Wash Solution I (try to do only 2, or at the most 3, at once to this point), vigorously plunge the rack up and down 1 minute without touching the bottom of the glass dish (to avoid breaking the slips and scratching the arrays).

8.  Individually transfer ships to slide dish with Wash Solution II.  Do no transfer the entire rack, sicne this will carry over too much SDS, which we are trying to get rid of.

9.  Place each array into a 50 ml conical tube and spin dry in the clinical centrifuge at 1000 rpm for 5 minutes (lowest rpm setting on the lab centrifuge).

10.  Try to scan array within a short period (hours) of completing the washing as the Cy dyes are unstable and may degrade differentially.                          

Scanning the Microarray with GenePix4000a Scanner Protocol

1. Turn on scanner, start Gene Pix Pro software.
2. Slide open scanner door; Insert chip hyb side down with label facing towards you.  Clip the chip holder easily around slide; do not push direcly down on the slip.
3. Choose the Hardware Settings window from the left menu.  Set photomultiplier tubes to 600 in both 635 nm (Cy5) and 532 nm (Cy3) channels. 
4. Perform a low resolution "preview scan".
5. Draw a scan area marquis around the entire array once you can see where all the spots are located.  Leave a small margin on all sides.
6. You can adjust the PMTs now to make the red and green images more equal; this will be done more carefully later too.
7. Under the hardware settings window, change the 'lines to average' to 2.  The scanner will now scan each pizel twice and average the data, reducing any background electronic noise.
8. Begin a high resolution 'DataScan' at lower speed.  As the image is scanning, go to the 'histogram' tab located at the top of the screen.  This tab will bring up a graph of the relative red and green intensities.  Histogram settings should be " Image: both  X Axis: fullscale, Y Axis: Log Axis On, Fullscale.  This histogram shows the percentage of normalized counts that are at a given intensity of signal.  It examines what you have selected (if zoomed, it only looks at the zoomed area, if dirt or artifacts are there, it includes them, etc.).  Try to select a clean area to use to reset the PMTs.  Make adjustments to the PMT settings so that the two curves appear to superimpose and if possible, neither one goes off scale.  Pizels with counts greater than 67,000 will be saturated and will be thrown out; pixels with data close to background will not give reproducible results.  Try for a representation of each color of pixels across the entire range, with the ratio as close to 1 as you can achieve (i.e. the two curves appear superimposed to the extent possible).
9. Stop the scan and begin the 'data scan' over the whole image to collect and store the image. This will take several minutes.
10. To save the image, go to the Open/Sae button and select "save images".
11. Save as type = Multi-image Tiff files, and include a date prefix (070303 for March 7, 2003) in the name, as well as an indication of the user and the gene or condition.  For example MY070303sir2x, for Michelle Yuen's data for the date indicated using the sir2 extra copy strain.  For the class  data, we will use the date scanned, bi164, and oxyrhythm in the name.  Save the wavelength 635 and the wavelength 532 images separately; if you wish you may also save the combined image as a JPEG file that would show nicely on your own computer at home.
12. Once the image is successfully scanned and saved, you can assign spot identities to the scanned spots.  We have prepared an excel file with the genes from ISB arrays in the same order that you will obtain the data from these scans.  It can be used by GenePix to assign the names as it determines the absorbances; it saves a tab-delimited text file of this information.  Or, you can use the scans and the program Scanalyze from Michael Eisen's laboratory to grid the image and obtain the absorbances. in a table.  This table can have the gene list copied to it for analysis.  If you want to put the Scanalyze results into GeneSpring, you need to take them into Excel, edit out the heading material up to the column headers, and then save as a text file.  That can be opened by GeneSpring using the Autoloader on the File Menu. 
13. You can import the output into GeneSpring for analysis, or you can import it into Excel.  We recommend both! 

Materials:

Gloves

Sterile tubes

Nutator

Micropipettors

Microfuge

Aluminum foil box

Racks for room temp incubation

Waterbath set at 42 degrees (need to be able to make it light tight).

Qiagen cleanup kit

Microcon 30 kit

Filter tips

Kimwipes

Corning microarray hyb chambers, 1 per student

microarrays, one per student

lifter cover slips, 2 per student and a couple of extra

Whoosh duster

NaBicarbonate pH9.0, 5 aliquots

DMSO in hood

95% ethanol for cleaning cover slips

MilliQ water, tube per student

Cy3 and Cy5 concentrated activated dyes, aliquotted  enough to label 20 ug of sample and dried in Speed Vac, taken from –20 degree freezer and put in ice bucket in foil coverings about 1 hr before lab (MY put in a freezer box for lab transport). Each student needs 2 tubes of Cy3 and 2 tubes of Cy5; give us one extra tube of each dye just in case.