Using Perkin Elmer Lifesciences Micromax TSA Labeling Kit

General Instructions

Indirect Labeling Tyramide Signal Amplification [link ppt file]

• The following protocol is an abbreviated version of the protocol that accompanies the kit. Read through the kit protocol before following the steps below
• Use RNase/DNase free reagents/supplies only. Wear gloves at all times
• Cy3/Cy5 dyes and reagents containing these dyes are light sensitive. Care should be taken to minimize exposure of these reagents to light

Determination of Labels and Amount of RNA Needed

1. Fluorescein label should be used for the untreated/control sample.

2. Biotin label should be used for the treated sample.
   
   One set of reactions (mL)

   Reagents	FL (Flourescein)	B (Biotin)
   2 mg total RNA in water	"x"	"x"
   1:10 diluted control RNA	5.0	5.0
   Reaction Mix Concentrate (Tube Y)	1.0	1.0
   Flourescein or Biotin Nucleotide	1.0	1.0
   RNase free water	13 – "x"	13-"x"
   Total	20.0	20.0

   
   
3. Incubate at 65°C for 10 minutes, then at 25°C for 5 minutes. 
4. Incubate at 42°C for 2-3 minutes. Add to each tube:
   
   2.5 µL RT Reaction Buffer (Tube A)
   2.0 µL AMV RT/RNase Inhibitor Mix (Tube E)
   
5. Incubate at 42°C for 60 minutes. Then, 4°C for 10 minutes. 
6. Add to each tube:
   
   2.5 µL 0.5 M EDTA (Mix well.)
   2.5 µL 1.0 N NaOH (Mix well by vortexing.)
   
7. Spin down at max speed for a few seconds. 
8. Incubate at 65°C for 30 minutes. Then, 4°C for 5 minutes, during which add 6.5 µL of 1M Tris HCl, pH 7.5, to each tube.

cDNA Purification

Fluorescein and biotin labeled cDNA should be purified in separate tubes. 

Use Millipore Microcon YM-100 Column (Catalog No. 42413):

1. Place membrane cartridge in a clean 1.5-mL Eppendorf tube.
2. Add 200 µL of 10 mM Tris, pH 7.5, to membrane cartridge. Add FL or B labeled reaction to the membrane cartridge.
3. Centrifuge the sample at 500 x g for 4 to 6 minutes, until sample volume reaches about 20 µL.
4. Add 400 µL of 10 mM Tris, pH 7.5, to membrane.
5. Centrifuge the sample at 500 x g for 4 to 6 minutes, until sample volume reaches about 20 µL.
6. Invert cartridge into clean 1.5-mL tube and centrifuge at 500 x g for 3 minutes.
7. Spin dry sample for 1-2 hours at 30°C in speed vac.
8. Resuspend sample in 20 µL hybridization buffer (Tube Q). Let stand at room temperature for 10 minutes. Remove 1 µL of each labeling reaction and dilute into 9 µL of TE buffer.

cDNA Analysis

1. Prepare serial dilutions of FL Control cDNA (Tube AA) and Biotin Control cDNA (Tube G).
   
   1:10 dilution (2 µL of Control cDNA into 18 µL of TE)
   1:20 dilution (5 µL of (A) into 5 µL of TE)
   1:40 dilution (5 µL of (B) into 5 µL of TE)
   1:80 dilution (5 µL of (C) into 5 µL of TE)
   
2. Prepare serial dilutions of the FL and Biotin labeled cDNA probes:
   
   1:10 (1 µL labeling reaction into 9 µL of TE)
   1:20 dilution (5 µL of (A) into 5 µL of TE)
   1:40 dilution (5 µL of (B) into 5 µL of TE)
   
3. With pencil, make boxes on the GeneScreen Membrane (Part F) for each dilution for the test and control cDNA.
4. Spot 1 µL of each dilution onto membrane in duplicates.
5. Wet membrane in 10 mL 2X SSC. Blot on filter paper.
6. Crosslink membrane using autocrosslink button on Stratagene UV Crosslinker.
7. Place membrane in a sealable pouch, and add 3.5 mL of TN Blocking Buffer with 10% goat serum (TNB-G). Heat seal. Incubate for 30 minutes on orbital shaker.
8. Cut open pouch, and add 20 µL each of Anti-FL-HRP Conjugate (Tube Z) and Streptavidin-HRP Conjugate (Tube J) into the pouch. Heat seal, and incubate for 30 minutes on orbital shaker.
9. Cut open pouch, and remove membrane. Place membrane in clean dish, containing 20 mL of TNT Buffer, and wash 4 times for 5 minutes each.
10. Make 4CN Plus Diluent working solution. Add 1 mL of 4CN Plus Diluent (Tube BB) into 9 mL of water. Add 200 µL of 4CN Plus Substrate (Tube CC). Use within 5 minutes of preparation.
11. Place membrane in a clean dish after washes. Add 4CN Plus Diluent working solution. Incubate in dark for 30 minutes. Check spots.

Hybridization

1. Heat probe mixture at 90°C for 2 minutes.
2. At room temperature, add probe mixture to array. Cover with coverslip. Avoid air bubbles.
3. Hybridize overnight at 60°C in water bath in hybridization with 2X SSC added to keep chamber moist.

Washes

These are all done at room temperature in a 50-mL conical tube at room temperature.

Wash 1

• 750 µL 20X SSC
• 30 µL 10% SDS
• Water to 30 mL

Wash just long enough for the coverslip to come off the slide.

Wash 2

• 750 µL 20X SSC
• 30 µL 10% SDS
• Water to 30 mL

Wash for 5 minutes.

Wash 3

• 90 µL 20 X SSC
• 30 µL 10% SDS
• Water to 30 mL

Wash for 5 minutes.

Wash 4

• 90 µL 20X SSC
• Water to 30 mL.

Wash for 2 minutes.

TSA Detection

1. Incubate array with 600 µL of TNB-10% Goat Serum (TNB-G) for 10 minutes at room temperature in hybridization chamber.
2. Wash slide in TNT Buffer for 1 minute.
3. Incubate for 10 minutes with 300 µL of Anti-FL-HRP conjugate solution (4 µL of Anti-FL-HRP Conjugate (Tube J) in 400 µL of TNB-G).
4. Wash 3 times for 1 minute each in TNT buffer.
5. Incubate for 10 minutes with 300 µL of Cyanine-3 Tyramide Solution (1 µL of Cyanine-3 Tyramide (Tube R) into 500 µL of Amplification Diluent (Tube S).
6. Wash 3 times for 5 minutes each in TNT buffer.
7. Incubate for 10 minutes with 300 µL of HRP Inactivation Solution (10 µL of 3 M Sodium Acetate (Tube U) into 290 µL of HRP Inactivation reagent (Tube T)).
8. Wash 3 times for 1 minute each in TNT buffer.
9. Incubate for 10 minutes with 300 µL of Streptavidin-HRP solution (4 µL of Streptavidin-HRP conjugate (Tube J) into 400 µL of TNB-G).
10. Wash 3 times for 1 minute each in TNT buffer.
11. Incubate for 10 minutes with 300 µL of Cyanine-5 Tyramide Solution (1 µL of Cyanine-5 Tyramide (Tube R) into 500 µL of Amplification Diluent (Tube S).
12. Wash 3 times for 5 minutes each in TNT buffer.
13. Wash for 1 minute in 0.06X SSC.
14. Spin dry and scan slide.