Reverse Transcriptase Labeling with Cy3 / Cy5 Worksheet

Date of labelling:  					    Name:  					
Slide ID number:  					    Print Date: 					

Put prehybridization solution at 42c to prewarm

	Cy3 label	Cy5 label	
Tissue			
Total or mRNA			
Date RNA prepared			

check 

Conc of RNA g/l			
  # of l used 			
 Amt of RNA (g)*			
* want 1- 2 g mRNA or 50-100 g total RNA per labeling reaction
Labelling
Cy3  	Cy5	Check as added  (Cy5 is very sensitive to fluorescent light so minimize the exposure)
 l  water	l  water
____   ____	1. Mix together:
  l  RNA	 l  RNA
____   ____         	RNA (or RNA + water)		8 l				
____   ____	 	oligo dT (18-21 mer, 2.5 g/l)		2 l	Date/source:	_______	
					Total volume	          10 l	
____   ____	2.  Heat 70c, 10 minutes
____   ____	3.  Place on ice for  5-10 minutes, then flash spin
4.  Prepare the labeling reaction cocktail:
Reagent	     		vol	[final]	    		Lot/stock # & Date
____   ____		5X Buffer (Gibco)		6 l	   1 X		____________________
____   ____		DTT (0.1M, Gibco)		3 l	 10 mM	____________________
____   ____		Low T’ dNTP’s (Sigma)	6 l	500 M dACG	____________________
(2.5 mM dACG, 1.0 mM dT)			200 M dT					
____   ____		Cy3- or Cy5-dUTP (1mM)	3 l	100 M	Cy3_________________
(FluoroLink Cy3-dUTP #53022 or Cy5-dUTP #55022)
(Amersham / Pharmacia)				Cy5_________________
____   ____		SuperScript II (200 U/l)	2 l	13 U/l	
Gibco  #18064-014				          SuperScript _______________
			Total volume			20 l per reaction			
____   ____	5.  Add a 20 l aliquot of the reaction cocktail to 10 l of RNA/oligo dT (total 30 l).
____   ____	6.  Finger flip and quick spin.
____   ____	7.  Incubate 42c, 1 hour.
____   ____	8.  Add 1 l SuperScript.  Continue incubation at 42c for another hour (2 hours total).

Prehybridize. Start soon after adding this second aliquot of SuperScript.
Note: Must use slides same day that you prehybridize them.
____ 1.  Place the slides in the plastic container (Corning CMT-GAPS slide container).
____ 2. Add 25 mls prewarmed (42c) hybridization buffer (5X SSC, 0.1% SDS and 1% BSA)
Incubate at 42 C for 45-60 min.  Mix periodically. 		Prehyb Date:_____________
RNase treatment
____   ____	9.  Add 1 l RNase A/H mix 	(0.5mg/ml RNase A, 1 U/l RNase H)    Date:__________
____   ____   10.  Incubate at 37c for 30 minutes to degrade RNA.					
Wash prehybrided slides  During the RNase treatment, wash slides and spin dry.
___1.  Remove container from water bath and wipe conatiner dry with kim wipe.
___2.  Remove slides and transfer to slide rack in 200-250 mls sterile nanopure water (roomtemp) by
 using forceps and gloved fingers (careful to avoid array area and only to touch edges of slides).
___3.  Once all slides are collected into rack, transfer the rack to second dish of clean water.
___4.  Wash the slides by dipping up/down several times or put on orbital shaker 5 minutes.
___5.  Transfer rack of slides to dish of 200-250 mls isopropanol (Fisher HPLC Grade # A451-1) 
___6.  Rinse in isopropanol by dipping up/down several times.
___7.  Spin dry at 500 rpm for 2 minute.
Notes: 		1. It is important to use the slides soon after the prehybridization. (within 1 to 2 hrs)
2. If only have one or two slides, might be more efficient to use 50ml conical tubes.

Post labeling clean up. Qiagen QIAquick PCR Purification kit (Cat No. 28104) Lot# _____________
       Note: All steps done at room temperature.
___ 1.  Place labeled spin column into collection tube.
___ 2.  Mix together desired Cy3-Cy5 probe pairs.
     	___ 3.  Add 320 l (5X probe volume) PB Buffer to labeled probe.
     	___ 4.  Transfer to column. Spin 11,000 rpm for Eppendorf 5417C for 1 min.
     	___ 5.  Discard flow through.
     	___ 6.  Add 750 l PE Buffer and spin 11,000 rpm for 1 minute.
     	___ 7.  Discard flow through. 
     	___ 8.  Place column back in collection tube and spin to remove traces of PE Buffer.
    	___ 9.  Transfer column to clean eppendorf tube. 
     	___10.  Add 50 l EB Buffer. Leave on bench one minute to dissolve.
     	___11.  Spin one minute to collect probe.
___12.  SpeedVac to desired volume (< 12 l) for the current size array (22x40mm cover slip) 
Takes about 17 minutes in the speed vac in Room 388 to reach 12 l.
Optional:  Store at –20c after step 11 or step 12, until ready to use.  Can be stored several days.

check

While probe is in SpeedVac, put 2X hybridization solution at 42c to prewarm

Prepare bench for hybridization. Prepare bench while waiting for probe to concentrate in SpeedVac.
___1.  Put 2x hyb solution (50% Formamide, 10X SSC, 0.2% SDS) at 42c (14 l/slide).	Date:________
___2.  Wipe down bench with water or alcohol to remove dust.
___3.  Put Poly A (10µg/ µl) on ice.						Date/Lot: ________________
___4.  Wipe and dust hybridization chambers and place parts on bench.
___5.  Put prehybridized slides into chambers without fastening. Label chambers with Sharpie.
___6.  Remove coverslips (22 x 40 mm), from package but leave on the plastic protection.
___7.  Place humidity papers (2/slide) on either parafilm or box lid and moisten with squirt bottle.
___8. Find round floater for placing samples in the hot water.
___9. Put 800 ml water in 1 liter Nalgene beaker and place in microwave. Turn on for 6 minutes.
 
Prepare probe. Water should be heating in microwave.
___1.  Retrieve probe from SpeedVac.
___2.  Add water if necessary to have 12 to 12.5 l volume.
	____ l probe 
	____ l water
___  3.  Add 1.5 l Poly A10 µg/ µl.  Finger flip. Quick spin.
	Total volume   ____  ml  (should be close to 14ml) 
___  4.  Place all tubes into round floater. Check to see that the water is boiling. 
___  5.  Carefully bring beaker of boiling water to bench.
___  6.  Place floater with tubes into freshly boiled water to denature at 95C for 3 min.
___  7. Spin one minute at highest speed. 
___  8.  Add an equal volume (14 ml) of prewarmed 2X hybridization buffer to the probe.
	Mix using pipetter but try not to make many bubbles.
	If using multiple samples add 2X hyb to all, put extras at 42c until ready to spot them.
___  9.  Remove plastic protection from a coverslip and pipette probe onto it.
___10. Working quickly, place slide (array facing down) onto droplet on cover slip 
	use pipette tip to gently press cover slip to work bubbles to edges.
___10.  Add saturated (not over saturated) ‘hydration papers’at end of slide, far from cover slip.
___11.  Assemble hybridization chamber quickly. Keep it level and avoid jerky movements.
___12.  Incubate for 16-20 hr at 42 C.

Prepare washes for tomorrow.  Need to do ahead of time so solutions are at correct temperature.
___ 1.  Take wash solutions out of the frige.
___ 2.  Wash Solution 1. Shake it well (SDS precipitates and settles at 4c) and:
	Pour about 200 mls into a large slide dish, cover, and put into 42c water bath.
	Pour about 200 mls into a small slide dish, add small metal slide rack, cover, put at 42c.
___ 3.  Wash Solutions 2 & 3:
	Leave on bench so will be room temperature tomorrow morning.

Washing of slides
Wash solution #1 (1X SSC, 0.2% SDS	Date______________ ):
	Should be ready to go (2 dishes of it at 42c since last night)
Wash solution #2 (0.2X SSC, 0.2% SDS	Date______________ ):
	Shake well to suspend SDS and fill small slide dish. Cover and label dish. Room temp.
Wash solution #3 (0.1X SSC			Date______________ ):
	Fill small slide dish. Cover and label dish. Room temp.

___1.  Remove hybridization cassette and wipe off excess water with kim wipe.
___2.  Disassemble hybridization cassette (using kimwipe to keep outside dry)
___3.  Quickly using your gloved fingers, remove humidity papers and place slide, cover slip side down,
 into the larger dish of Wash Solution #1 in 42c until cover slip gently slides off (avoid cover slip scratching the array). 
___4. Once cover slip slides off, transfer slide to the slide rack that is in the second dish of Wash
Solution #1 at 42c. Repeat using same solution for all slides that need washing.
___5.  Once all slides are collected in Wash #1, move this dish to the orbital shaker, cover with
aluminum foil to minimize exposure to light, and shake for 5 minutes.
___6.  Transfer rack to Wash #2 at room temperature and shake for 5 minutes.
___7.  Transfer rack to Wash #3 at room temperature and shake for 5 minutes.
___5.   Spin dry at 500 rpm for 2 minutes, 25c.

Ready to scan!

Scan worksheet
In order to avoid confusion between scans, please fill out the table underneath as you perform scans. This will also make your images readily available for quantification.

Print ID: ______		Slide letter-number: ________		
	(e.g. MP0011)					(e.g. D09)
Brief description of the experiment:

Channel 1	Channel 2	
Scan A   Scanarray___  GenePix___ (check)

      Date (mm/dd/yy):________
      Time(hh:mm):_______ AM PM (circle one)

      Laser power:_____%
      PMT:_____% (if Scanarray)
               _____V (if GenePix)

   Construct file name:
            (e.g. MS_0011_D09_1_A)
	Scan A   Scanarray___  GenePix___ (check)

       Date (mm/dd/yy):________
       Time(hh:mm):_______ AM PM (circle one)

       Laser power:_____%
       PMT:_____% (if Scanarray)
                 _____V (if GenePix)

   Construct file name:
            (e.g. MS_0011_D09_2_A)	
Scan B   Scanarray___  GenePix___ (check)

      Date (mm/dd/yy):________
      Time(hh:mm):_______ AM PM (circle one)

      Laser power:_____%
      PMT:_____% (if Scanarray)
                _____V (if GenePix)

   Construct file name:
            (e.g. MS_0011_D09_1_B)
	Scan B   Scanarray___  GenePix___ (check)

      Date (mm/dd/yy):________
      Time(hh:mm):_______ AM PM (circle one)

      Laser power:_____%
      PMT:_____% (if Scanarray)
                _____V (if GenePix)

   Construct file name:
            (e.g. MS_0011_D09_2_B)
	
Scan C   Scanarray___  GenePix___ (check)

      Date (mm/dd/yy):________
      Time(hh:mm):_______ AM PM (circle one)

      Laser power:_____% 
      PMT:_____% (if Scanarray)
               _____V (if GenePix)

   Construct file name:
            (e.g. MS_0011_D09_1_C)
	Scan C   Scanarray___  GenePix___ (check)

       Date (mm/dd/yy):________
       Time(hh:mm):_______ AM PM (circle one)

       Laser power:_____% 
       PMT:_____% (if Scanarray)
                _____V (if GenePix)

   Construct file name:
            (e.g. MS_0011_D09_2_C)