Liu Lab protocol

Bruno Fang/Jacy Villa

Updated by Amir Jazaeri

November 1, 2001

Jazaeria@mail.nih.gov

Protocol for Amplification of RNA –Modified from Eberwine

Version 1.4 Nov. 2001

Reagents

• Superscript II RT, Gibco Life technologies # 18064-014
• E. coli DNA Polymerase I, Gibco Life technologies # 18010-025
• E. coli DNA Ligase, Gibco Life technologies # 18052-019
• E. coli RNAse H, Gibco Life technologies # 18021-014
• T4 DNA Polymerase, Gibco Life technologies # 18005-025
• 5X second strand buffer, Gibco Life technologies # 10812-014
• dNTP Set, 10mM Gibco Life
• T7-(dT)24 Primer, GENSET Corp), HPLC purified DNA-5’-GGC-CAG-TGA-ATT-GTA-ATA-CGA-CTC-ACT-ATA-GGG-AGG-CGG-(dT)24-3’
• DEPC treated water, Ambion #9902 or Water, Molecular Biology Grade, BioWhittaker, #16-001Y
• RNAsin, Promega #N2515
• EDTA 0.5 M
• NaOH 1 M
• Phase Lock gel, 5 prime to 3 prime, Inc #pl-188233 for 200 or #pl-175850 for 50
• Phenol/Chloroform/isoamyl alcohol, Ambion #9732
• 7.5M Ammonium Acetate, Sigma #A2706
• 95% Ethanol
• 80% Ethanol
• Linear acrylamide, Ambion #9520
• T7 Megascript Kit, Ambion #1334
• Random hexamers, Gibco Life technologies #48190-011
• Qiagen Rneasy Mini Kit (50 samples) #74104

Check Before Starting

• Heating Block at 70ºC
• 95% and 80% ethanol in -20ºC
• Cold bath at 16ºC

First Strand Synthesis

1. Primer hybridization:

· 1-3 mg Total RNA

· (Variable) Nuclease free H₂O (to complete 1^st strand final reaction volume of 20 ml)

· 1 ml T7-(dT)₂₄ primer (2µg/ml)

2. Incubate at 70ºC for 10 minutes
3. Quick spin and put on ice
4. Add:
• 4 ml 5X First strand cDNA buffer
• 2 ml 0.1M DTT
• 2ml 10mM dNTP mix
• 1ml RNAsin
5. Add 2 ml Superscript II
6. Mix well and incubate at 42ºC for 1 hour
7. Centrifuge briefly, place on ice

Second Strand Synthesis

8. Add:

· 91 ml DEPC treated H₂O

· 30 ml Second strand buffer

· 3 ml 10 mM dNTP mix

· 4 ml DNA Polymerase I (10U/ ml)

· 1 ml DNA Ligase (10U/ml)

· 1 ml RNAse H (2U/ml)

Final Volume: 150 ml

9. Gently tap tube to mix, briefly centrifuge
10. Incubate at 16ºC for 2 hours
11. Add 2 ml (10U) T4 DNA Polymerase
12. Cool for 5 minutes at 16ºC
13. Stop reaction with 10 ml of 0.5 M EDTA, then add 10 ml of 1M NaOH
14. Incubate at 65ºC for 10 minutes, then neutralize solution with 25 ml Tris-HCl (pH=7.5)

Clean Up of Double Stranded cDNA

15. Pellet the Phase Lock Gel in a microcentrifuge at maximum speed for 30 seconds
16. Add 198 ml (equal volume) of (25:24:1) Phenol : chloroform : isoamyl alcohol (saturated with 10 mM Tris-HCL pH 8.0/1mM EDTA) to the final DNA synthesis preparation (198ml) to a final volume of 396 ml. Mix well by pipetting up & down vigorously.
17. Transfer the entire cDNA-phenol/chloroform mixture to the PLG tube
18. Do not vortex. Microcentrifuge at maximum speed for 2 minutes
19. Transfer the aqueous supernatant to a new 1.5 ml tube
20. Add 1 ml linear acrylamide
21. Add 0.5 volumes of 7.5M Ammonium Acetate + 2.5 volumes (include the added Ammonium Acetate) of 95% ethanol stored at –20 to the sample and vortex. (may stop here, keep sample at -20ºC and continue next day)
22. Centrifuge at maximum speed in a microcentrifuge at room temperature for 20 minutes
23. Remove supernatant. Wash pellet (may be invisible) with 0.5 ml of 80% ethanol
24. Centrifuge at maximum speed for 5 minutes at room temperature
25. Carefully pour-off the 80% ethanol.
26. Repeat the 80% ethanol wash once again
27. Air dry the pellet (~ 15 min.)
28. Resuspend the pellet in 16 ml of Nuclease-free water

In Vitro Transcription

29. Ambion T7 Megascript kit- Follow manufacturer’s instructions for a total 40 ml reaction (double the 20 ml standard reaction, 37ºC for 4-5 hours)

SET UP REACTION AT ROOM TEMPERATURE

If done on ice, spermidine in the buffer can lead to template precipitation

• 16µl of template DS DNA (from step 28).
• 4µl of 10x Reaction Buffer
• 4µl of ATP solution (75mM T7)
• 4µl of CTP solution (75mM T7)
• 4µl of GTP solution (75mM T7)
• 4µl of UTP solution (75mM T7)
• 4µl of Enzyme Mix
• Incubate at 37ºC for 5-6 hours.

30. Add 60 ml Nuclease free water to bring total volume up to 100ml.
31. Follow “RNA clean-up” protocol in the Qiagen RNeasy mini handbook
32. Elute with 30ml of Nuclease free water.
33. Check O.D. and ratio

Expected yield: ~10 X starting amount of total RNA

34. Proceed using the Liu lab protocol for generating probe for microarrays using total RNA. Use 3 to 5 mg amplified RNA and random hexamer primers (2 ml of 3 mg/ml instead of oligo-dT primer)

For 2^nd Round Amplifications:

• Ressuspend 0.5-1.0mg of amplified RNA in 11 ml ultrapure water

First Strand Synthesis

1. Add 1 ml Random hexamer (1 mg/ml)
2. Incubate 70ºC for 10 minutes, then chill on ice
3. Equilibrate at room temperature for 10 minutes
4. Add:
• 4 ml 5X First strand cDNA buffer
• 2 ml 0.1M DTT
• 2ml 10mM dNTP mix
• 1ml RNAsin
5. Mix and incubate at 42ºC for 2 minutes
6. Add 2 ml Superscript II
7. Mix well and incubate at 42ºC for 1 hour
8. Add 1 ml RNAse H and incubate at 37ºC for 20 minutes
9. Heat to 95ºC for 2 minutes and chill on ice

Second Strand Synthesis

10. Add 1 ml T7-oligodT primer (0.5 mg/ml), incubate 70ºC for 5 minutes and at 42ºC for 10 minutes
11. Then add:

· 91 ml DEPC treated H₂O

· 30 ml Second strand buffer

· 3 ml 10 mM dNTP mix

· 4 ml DNA Polymerase I (10U/ ml)

· 1 ml DNA Ligase (10U/ml)

· 1 ml RNAse H (2U/ml)

Final Volume: 150 ml

12. Gently tap tube to mix, briefly centrifuge
13. Incubate at 16ºC for 2 hours
14. Add 2 ml (10U) T4 DNA Polymerase
15. Cool for 5 minutes at 16ºC
16. Stop reaction with 10 ml of 0.5 M EDTA, then add 10 ml of 1M NaOH
17. Incubate at 65ºC for 10 minutes, then neutralize solution with 25 ml Tris-HCl (pH=7.5)

Clean Up of Double Stranded cDNA and In Vitro Transcription: Proceed as above