See the Gene List for the RT² Profiler Human Angiogenesis PCR Array.

Storage Conditions:  The kit is shipped on dry ice or cold packs. For long-term storage, keep at -20ºC. If entire volume is not be used all at once, divide into aliquots and store at -20ºC. Avoid repeated freezing and thawing.

Shelf Life: All reagents are stable for 6 months after receipt of the kit if stored at the recommended temperature.

AmpH-024: One (1) tube containing 90µl RT² PreAMP cDNA Human Angiogenesis Primer Mix solution
                Enough for 12 25-µl standard PreAMP reactions

1. Ensure that you do not contaminate the RT² PreAMP cDNA Synthesis Primer Mix by using a fresh pipette tip every time you draw an aliquot from the tube.

2. Perform first strand synthesis using the RT² PreAMP cDNA Synthesis Kit as follows:
   
   A. Add 2 µl of GE to 8 µl of RNA (1-100ng). Incubate at 42°C for 5 min and immediately chill on ice.
   B. Mix a master mix for the RT reaction as below:

   	For 1 reaction
   BC3	4 µl
   RE	1 µl
   RI	1 µl
   P2	1 µl
   RNase/DNase free H2O	3 µl
   Total	10 µl

   C. Add 10 µl of the RT master mix to 10 µl GE-treated RNA.
   D. Incubate at 42ºC for 30 min and heat at 95ºC for 5 min. Chill on ice or store at -20ºC until proceeding to the next step.

3. For the normal standard reaction, mix the following components in a PCR tube:

   12.5	µl	2X RT² PreAMP PCR Master Mix
   7.5	µl	RT² PreAMP cDNA Primer Mix specific for the RT² Profiler™ PCR Array of your choice
   5.0	µl	Template undiluted cDNA from the 20-µl first strand synthesis reaction (step 2D)
   25.0	µl	final volume

4. Perform 12 cycles of PCR in a thermal cycler:
   NOTE: The 10 min step at 95ºC is required to activate the HotStart Taq DNA polymerase.
   95ºC, 10 min; 12 cycles of (95ºC, 15 sec; and 60ºC, 2 min); 4ºC forever
5. Add 2 µl of the Side Reaction Reducer to each pre-amplified reaction, and incubate at 37ºC for 15 min followed by heat inactivation at 95ºC for 5 min.
6. Dilute the 27-µl pre-amplified templates to 111 µl by adding 84 µl of dd H₂O. For immediate use, keep on ice prior to loading onto the RT² Profiler™ PCR Array, or store at -20ºC until ready.
7. For use in the RT² Profiler™ PCR Array, prepare the Experimental Cocktail with the following components in 15-mL conical tube:

   1275	µl	2X SABiosciences RT² qPCR SYBR Green Master Mix (Note: Use the appropriate master mix specific for your real-time PCR instrument)
   102	µl	diluted PreAMP PCR reaction
   1173	µl	ddH2O
   2550	µl	final volume

8. Add 25 µl of the above Experimental Cocktail to each well of the PCR Array, preferably from a reservoir with an eight-channel pipettor (or a twelve-channel pipettor but only using eight tips).
9. Run the following real-time thermal cycler program:
   NOTE: The 10 min step at 95ºC is required to activate the HotStart Taq DNA polymerase.
   95ºC, 10 min; 40 cycles of (95ºC, 15 sec; and 60ºC, 60 sec)
10. Program the real-time thermal cycler to detect and record the SYBR® Green I signal from every reaction during the annealing step of each cycle.

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