DiDonato, Ray 1-3
RNA Amplification with Message AmpII-Bacteria Kit
Amplification of 200-500 ng of total RNA
In a 0.2 ml PCR tube, bring 200-500 ng total RNA up to a total volume of 5 l.
Heat at 70°C 10 minutes in a thermocycler
Place on ice for 3 min.
Make Polyadenylation Master Mix
a. Combine for each amplification tube:
i. 1.65 l nuclease-free H2O
ii. l 10X Poly(A) Tailing Buffer
iii. l RNAse Inhibitor
iv. 0.55 l Poly(A) Tailing ATP
v. l PAP
b. Mix with gentle pipetting up and down
5. Add 5 l of Polyadenylation Master Mix to each tube
6. Mix reaction by gentle pipetting
7. Incubate in a thermocycler at 37°C for 15 minutes.
8. Place on ice
9. Make Reverse Transcription Master Mix
a. Combine for each amplification tube:
i. 3.3 l nuclease-free H2O
ii. l T7 Oligo(dT) VN
iii. l 10X First Strand Buffer
iv. 4.1 l dNTP Mix
v. l ArrayScript
b. Mix with gentle pipetting up and down
10. Add 10 l Reverse Transcription Master Mix to each tube
11. Mix reaction with gentle pipetting up and down
12. Incubate for 2 hours at 42°C in a thermocycler.
13. In last 5 minutes of incubation, prepare the Second Strand Master Mix (on ice):
a. Combine for each amplification tube:
i. 69.3 l nuclease-free H2O
ii. 11.0 l 10X Second Strand Buffer
1. Vortex well to resuspend any precipitate before adding
iii. 4.4 l dNTP mix
iv. 2.2 l DNA Polymerase
v. l RNase H
b. Mix with gentle pipetting up and down
14. Place tubes on ice
15. Add 80 l Second Strand Master Mix
16. Gently pipet up and down to mix
17. Place in 16°C thermocycler with the heated lid turned off for 2 hours.
18. Preheat nuclease-free water to 50°C for at least 10 minutes
a. need 20 l per amplification
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DiDonato, Ray 2-3
19. Assemble a cDNA Filter Cartridge into a collection tube for each amplification
reaction.
20. Transfer the reaction to a 1.5 ml screw-cap tube
21. Add 250 l cDNA Binding Buffer
22. Mix by gentle vortexing.
23. Pipet sample onto cDNA Filter Cartridge
24. Spin for 1 minute at 10,000 x g (~10,6000 rpm).
25. Add 500 l Wash Buffer (ethanol added) to cDNA Filter Cartridge
26. Spin 1 minute at 10,000 x g
27. Discard flow-through
28. Spin for an additional minute at 10,000 x g to get rid of residual ethanol.
29. Transfer cDNA Filter Cartridge to a new collection tube
30. Add 20 l preheated nuclease-free H2O
31. Incubate for 2 minutes at room temperature.
32. Spin 1.5 minutes at 10,000 x g
33. Place on ice
34. Make an IVT (In vitro Transcription) Master Mix:
a. Combine for each amplification tube:
i. 4.4 l T7 ATP Soln (75mM)
ii. 4.4 l T7 CTP Soln (75 mM)
iii. 4.4 l T7 GTP Soln (75 mM)
iv. 4.4 l T7 UTP Soln (75 mM)
v. 4.4 l 10X T7 Reaction Buffer
vi. 4.4 l T7 Enzyme Mix
b. Mix by gentle pipetting up and down
35. In a fresh PCR tube mix:
a. 16 l cDNA
b. 24 l IVT Master Mix
c. Pipet gently up and down to mix.
36. Incubate 14 hours at 37°C in a thermocycler, followed by 4°C forever.
37. Preheat nuclease-free H2O for at least 10 minutes at 50°C.
38. Assemble an aRNA Filter Cartridge and collection tube for each amplification.
39. Add 60 l nuclease-free H2O to each amplification
40. If you notice a semi-solid precipitate, gently vortex then pipet up and down.
41. Transfer to a 1.5 ml screw-cap tube.
42. Add 350 l aRNA Binding Buffer
43. Vortex gently to mix.
44. Add 250 l 100% EtOH (ACS Grade).
45. Mix by pipetting up and down 3 times
46. Transfer entire mix to aRNA Filter Cartridge.
47. Spin 1 minute at 10,000xg.
48. Discard flow-through.
49. Add 650 l Wash Solution
50. Spin 1 minute at 10,000xg.
51. Discard flow-through
DiDonato, Ray 3-3
52. Spin for an additional minute at 10,000xg to get rid of residual EtOH.
53. Add 75 l preheated nuclease-free H2O
54. Incubate 2 minutes at room temperature
55. Spin at 10,000 x g for 1.5 minutes.
56. Repeat steps 53-55
57. Place aRNA tube on ice
58. Measure the A260 using the NanoDrop for concentration.
a. Typically get 160-180 g from 500 ng starting RNA
b. Consult manual for expected yields for other starting amounts.
59. Precipitate aRNA after purification.
60. For some reason, aRNA incorporates label much better if it is precipitated first.
61. Probably something in the MessageAmp reagents makes it into the final aRNA
and reduces labeling efficiency
62. Add 15 l NH4OAc and 495 l 95% EtOH to each tube.
63. Vortex to mix
64. Place at -20°C for at least 1 hour.
65. Spin precipitated aRNA at full speed for 30 minutes at 4°C.
66. Wash with 75% EtOH
a. Prepare this from the 95% stock only
b. Do Not prepare from the 100% stock, as the drying agent in this is a
problem
67. Repeat step 66 2 times
68. Air-dry
69. Resuspend to a concentration of ~1 g/l.
70. Store at -20°C.
71. Proceed to RNA Labeling in Microarray Core SOP