Ambion and Mouser, Paula 1-3
mRNA Amplification Procedure
Use the Ambion MessageAmp II-Bacteria Kit, cat #1790
Use the Ambion web site (www.ambion.com/tools.ma2bact) to calculate master mix
volumes.
Day 1:
1.
2.
3.
4.
5.
6.
7.
With new kit, add 24mL 100% ethanol to wash buffer.
Clean surfaces, pipetmen, tips, etc with Bleach, Ethanol, and then RNaway.
Bring 500 ng RNA up to 5uL using Nuclease-Free water
Incubate for 10 minutes at 70°C in thermocycler
Centrifuge briefly (~5 seconds)
Place on ice for 3 minutes
Make polyadenylation cocktail at room temperature:
a. Calculate adjustment for mix below by X number of samples + 5%
b. Mix used for each sample:
i. 1.5ul Nuclease-Free water
ii. 1.0ul 10x poly(a) tailing buffer
iii. 1.0ul RNase Inhibitor
iv. 0.5ul poly(a)tailing ATP
v. 1.0ul PAP
c. Gently vortex
d. Centrifuge ~5 seconds to collect mix at bottom of tube
8. Add 5ul polyadenylation mix to each sample
9. Mix by gentle vortexing
10. Collect liquids with a quick spin
11. Incubate for 15 minutes at 37°C in thermocycler (don’t use heated lid)
12. Place reactions on ice
13. Make reverse transcription cocktail at room temperature:
a. Calculate adjustment for mix below by X number of samples + 5%
b. Mix used for each sample:
i. 3.0ul Nuclease-Free water
ii. 1.0ul T7 oligo(dT) VN
iii. 1.0ul 10X First Strand Buffer
iv. 4.0ul dNTP Mix
v. 1.0ul ArrayScript
c. Gently vortex
d. Centrifuge ~5 seconds to collect mix at bottom of tube
14. Add 10ul reverse transcription mix to each sample
15. Mix by gentle vortexing
16. Collect liquids with a quick spin
17. Incubate for 2 hours at 42°C
18. Place reactions on ice
19. Make second strand cocktail on ice:
a. Calculate adjustment for mix below by X number of samples + 5%
b. Mix used for each sample:
Ambion and Mouser, Paula 2-3
i. 63ul Nuclease-Free water
ii. 10ul 10X Second strand buffer
iii. 4ul dNTP Mix
iv. 2ul DNA Polymerase
v. 1ul RNase H
c. Gently vortex
d. Centrifuge ~5 seconds to collect mix at bottom of tube
20. Add 80ul of second strand mix to each reaction.
21. Incubate for 2 hours in pre-cooled 16°C thermal cycler
a. Don’t use heated lid.
22. Preheat 10ml bottle Nuclease-Free water to 50°C for 10 minutes
23. Warm cDNA binding buffer to 37°C for 15 minutes
24. Add 250ul cDNA binding buffer
25. Mix by vortexing
26. Pipet onto cDNA filter column
27. Centrifuge for 1 minute at 10,000 rpm at room temperature.
28. Discard flow-through.
29. Add 500ul wash buffer to column.
30. Centrifuge for 1 minute at 10,000 g at room temperature.
31. Discard flow-through.
32. Spin an additional 1 minute to remove all ethanol.
33. Transfer column to clean cDNA elution tube
34. Apply 20ul preheated 50°C Nuclease-Free water to column.
35. Leave at room temperature for 2 minutes.
36. Spin for 1.5 minutes at 10,000 rpm.
37. Make IVT mix at room temperature:
a. Calculate adjustment for mix below by X number of samples + 5%
b. Mix used for each sample:
i. 4ul T7 ATP Solution
ii. 4ul T7 CTP Solution
iii. 4ul T7 GTP Solution
iv. 4ul T7 UTP Solution
v. 4ul 10X T7 Reaction Buffer
vi. 4ul T7 Enzyme Mix
c. Gently vortex
d. Centrifuge ~5 seconds to collect mix at bottom of tube
38. Add 24ul IVT master mix to purified cDNA
39. Incubate for 14 hours at 37°C
a. Finished for day 1
Day 2:
1.
2.
3.
4.
5.
Preheat 10ml bottle Nuclease-Free water to 50°C for 10 minutes
Warm aRNA binding buffer to 37°C for 15 minutes
Add 60ul Nuclease-Free water to incubated reaction (to bring to 100 ul)
Add 350ul aDNA binding buffer to each sample
Mix by gentle vortexing
Ambion and Mouser, Paula 3-3
6. Add 250ul 100% Ethanol
7. Pipet 3 times to mix
a. Don’t vortex this step!
8. Pipet onto aRNA filter column
9. Centrifuge for 1 minute at 10,000 rpm at room temperature.
10. Discard flow-through.
11. Place filter in new tube.
12. Add 650ul wash buffer to aRNA column
13. Centrifuge for 1 minute at 10,000 g
14. Discard flow-through.
15. Centrifuge an additional 1 minute to remove ethanol.
16. Place filter in new clean tube.
17. Add 75ul preheated 50°C Nuclease-Free Water to filter.
18. Leave at room temp for 2 minutes.
19. Centrifuge for 1.5 minutes for 10,000 rpm.
20. Repeat elution with additional 75ul water.
21. Quantify concentration on nanodrop.
Precipitation and Resuspension for Microarray Analysis:
1. Add 1/10 volume 5M NH4OAc to the purified aRNA.
a. i.e. for an eluted 150ul sample, this is 15ul
2. Add 2.5 volumes of 100% Ethanol
a. i.e. for an eluted 150ul sample, this is 415ul.
3. Mix well
4. Incubate at -20°C for a minimum of 30 minutes
5. Centrifuge on high (14,000 rpm) at 4°C for 15 minutes
6. Remove supernatant.
7. Wash pellet with 500ul 70% cold ethanol
8. Centrifuge on high (14,000 rpm) at 4°C for 10 minutes
9. Remove ethanol.
10. Dry out pellet for 8 minutes at room temperature.
11. Resuspend in desired volume of DEPC water.
a. Should be at 1 ug/ul concentration, with a minimum 20 ug.