PROTOCOL FOR SINGLE CELL mRNA AMPLIFICATION/ REVERSE NORTHERN ANALYSIS
Yeh Lab Version 1.1
C. Spencer
10-5-98
References:
1. J. Eberwine, H. Yeh, K. Miyashiro, Y. Cao, S. Nair, R. Finnell, M. Zettel and P. Coleman. PNAS 89: 3010-3014 (1992).
2. J. Eberwine. Biotechniques 20: 584-591 (1996).
I. First round amplification using magnetic bead support
A. Preparation of T7-oligo-d(T)24-linked beads
1. Briefly vortex the beads in storage buffer.
2. Transfer 2.5µl (=250ng) to a new tube.
3. Add 10µl of unlinked beads as a carrier.
4. Add 100µl TE, vortex briefly, then place tube in magnetic holder for 30 sec.
Alternatively, you may wish to pellet the beads by a quick spin and then place the tube in magnetic holder to remove solution.
5. While tube is in holder, remove solution.
6. Add another 100µl TE, vortex briefly, then place tube in magnetic holder for 30 sec.
7. Remove wash solution.
8. To the beads, add
DEPC-H2O24 µl
10x RT buffer 5 µl
9. Keep beads on ice, or at 4 °C, until use.
B. First strand cDNA synthesis
10. Add to beads in RT buffer: (29 µl)
Cell contents plus additional DEPC-H2O10 µl
4-dNTPs (2.5mM each) 5 µl
100mM dithiothreitol (DTT) 4 µl
RNasin 0.5 µl
AMV-reverse transcriptase (25units/µl) 1 µl (25U final)
Final volume:50 µl
For fixed tissue, add 0.5µl of 10% digitonin in ddH2O (0.1% final concentration).
11. Mix gently; incubate at 42 °C for 60-90 min.
At this point, the beads could be stored at 4 °C (for a few days) or in the freezer (for weeks) until ready to proceed with several samples. Try to avoid repeated freeze-thaw cycles.
C. Second strand cDNA synthesis
12. Centrifuge briefly to bring down condensation.
13. Heat at 90-95 °C for 3 min to denature RNA:DNA hybrid.
14. Cool quickly on ice; centrifuge briefly to bring down condensation.
15. Place tube(s) in magnetic holder; remove solution.
First strand cDNA is now linked to the beads.
16. Wash the beads once in 100µl 1x 2nd strand buffer; remove wash solution while tube is in the magnetic holder.
17. Add to the magnetic beads:
DEPC-H2O28.3 µl
10x 2nd strand buffer 4 µl
100mM DTT* 2 µl
4 dNTPs (2.5mM each) 4 µl
random hexamers (100ng/µl) 1 µl
T4 DNA polymerase (5U/µl) 0.2 µl (1U final)
Klenow (5U/µl) 0.5 µl (2U final)
Final volume: 40 µl
* replace with DEPC-H20 if already included in the 10x 2nd strand buffer
18. Mix gently; incubate at 14 °C overnight.
19. Centrifuge briefly to bring down condensation.
20. Wash 2 times with 100µl of TE buffer.
21. Wash once with 100µl of 1x RNA amplification buffer; remove wash solution.
D. First round aRNA amplification (cold reaction)
22. Add to the magnetic beads:
DEPC-H2O11.5 µl
10x RNA amplification buffer 2 µl
20mM spermidine* 2 µl
4 NTPs (with UTP) (2.5mM each) 2 µl
100mM DTT 1 µl
RNasin 0.5 µl
T7 RNA polymerase (1000U/µl) 1 µl (1000U final)
Final volume: 20 µl
* replace with DEPC-H20 if already included in the 10x RNA amplification buffer
23. Mix gently; incubate at 37 °C for 4 hours.
Increased RNA synthesis has been observed by increasing the incubation time to 6 hrs, with a slight additional increase between 6 and 8 hrs. After 8 hrs, RNA degradation becomes a significant factor.
24. Centrifuge briefly to bring down condensation.
25. Place tube in magnetic holder; remove and transfer solution containing aRNA to a new tube. *** SAVE the SOLUTION! ***
26. Add 20µl TE to the beads and briefly vortex.
27. Place tube in magnetic holder; remove and transfer solution to tube containing aRNA.
The beads may be stored at 4 °C or -20 °C in TE for future use.
28. Phenol-chloroform extract aRNA. Add to tube containing aRNA-
DEPC-H2O 95 µl
3M ammonium acetate* 15 µl
chloroform 75 µl
buffer-saturated phenol 75 µl
Final volume:300 µl
*Ammonium acetate is more efficient than sodium acetate at removing free NTPs. Adding salt at this step (instead of the subsequent ethanol step) improves separation of the aqueous and organic phases.
29. Vortex for 10 sec. Centrifuge for 3 min. Carefully remove 145µl aqueous (top) phase to a new tube.
30. Precipitate with ethanol. Add
tRNA* (1µg/µl) 1 µl
100% ice-cold ethanol450 µl
*Use of a carrier is highly recommended to aid precipitation of miniscule amounts of RNA (esp. from single cells). However, tRNA can sometimes cause artefacts in PCR reactions. While this is usually not a problem with routine PCR analyses, it may be critical when doing differential display-PCR, for example. Glycogen is also thought to interfere with many manipulations. Other carriers have not been tested, but may be appropriate in these cases.
31. Leave at -20 °C for at least one hour. Centrifuge at 18,000 rpm at 4 °C for 30 min. Resuspend the pellet in 18.5µl of DEPC-H2O.
II. Conversion of aRNA to double-stranded cDNA
A. First strand cDNA synthesis
32. Denature 18.5µl aRNA sample at 90- 95 °C for 3 min.
33. Cool quickly on ice; centrifuge briefly to bring down condensation.
34. Add to denatured aRNA sample: (18.5 µl)
10x RT buffer 3.0 µl
4 dNTPs (2.5mM each) 3.0 µl
random hexamers (100ng/µl) 1.0 µl
100mM DTT 2.0 µl
RNasin 0.5 µl
AMV reverse transcriptase (25U/µl) 1.5 µl (approx. 40 units)
Final volume:30.0 µl
35. Mix gently; incubate at 42 °C for 90 min.
36. Phenol-chloroform extract ss-cDNA. Add
DEPC-H2O 105 µl
3M sodium acetate 15 µl
chloroform 75 µl
buffer-saturated phenol 75 µl
Final volume: 300 µl
37. Vortex for 10 sec. Centrifuge for 3 min. Carefully transfer 145µl aqueous (top) phase to a new tube.
38. Add 300µl 100% ice-cold ethanol to precipitate.
39. Leave at -20 °C at least one hour. Centrifuge at 18,000 rpm at 4 °C for 30 min. Resuspend the pellet in 12.3µl of DEPC-H2O.
B. Second strand cDNA synthesis
40. Heat at 90-95 °C for 3 min to denature aRNA:DNA hybrid.
41. Cool quickly on ice; centrifuge briefly to bring down condensation.
42. Add to sample: (12.3 µl)
10x KFI buffer 2 µl
100mM DTT* 2 µl
4 dNTPs (2.5mM each) 2 µl
T7- oligo(dT)24primer (100ng/µl) 1 µl
T4 DNA polymerase (5U/µl) 0.2 µl (1U final)
Klenow (5U/µl) 0.5 µl (2U final)
Final volume: 20 µl
* replace with DEPC-H20 if already included in the 10x KFI buffer
43. Mix gently; incubate at 14 °C overnight.
44. Centrifuge briefly to bring down condensation.
C. Blunt-end reaction (optional)
If planning to do reverse Northerns, you may wish to blunt-end to eliminate possible “wrap-around” RNA synthesis, which could reduce (via RNA-RNA self-hybridization) the amount of aRNA probe available for hybridization to cDNAs on the blot.
45. Add to 2nd strand reaction: (20 µl)
DEPC-H2O 21 µl
10x KFI buffer 3 µl
100mM DTT* 3 µl
4 dNTPs (2.5mM each) 3 µl
T4 DNA polymerase (5U/µl)0.2 µl
Final volume:50 µl
* replace with DEPC-H20 if already included in the 10x KFI buffer
46. Incubate at 37 °C for 15-30 min.
47. Phenol-chloroform extract ds-cDNA. Add
DEPC-H2O 85 µl (115 µl if not blunt-ending)
3M sodium acetate 15 µl
chloroform 75 µl
buffer-saturated phenol 75 µl
Final volume: 300 µl
48. Vortex for 10 sec. Centrifuge for 3 min. Carefully transfer 145µl aqueous (top) phase to a new tube.
49. Add 300µl 100% ice-cold ethanol to precipitate.
50. Leave at -20 °C at least one hour. Centrifuge at 18,000 rpm at 4 °C for 30 min. Resuspend the pellet in 20µl of DEPC-H2O.
At this point the sample may be used for PCR analysis in a 1:100 final dilution.
III. Second round aRNA amplification (hot reaction)
51. Drop-dialyze 10µl sample against 50ml DEPC-H2O for at least 4 hours.
52. Combine:
1/10th of dialyzed sample plus add’l DEPC-H2O7.7 µl
10x RNA amplification buffer 2.0 µl
20mM spermidine* 2.0 µl
100mM DTT 1.0 µl
3 NTPs (ATP, GTP, UTP) (2.5mM each)2.0 µl
100µM CTP0.8 µl
RNasin 0.5 µl
-[32P]-CTP (3000Ci/mmol; 1mCi/100µl)3.0 µl (4mM final = 80pmol)
-remove 0.5µl and spot onto 1MM Whatman paper (= “TCA-before”)
add T7 RNA polymerase (1000U/µl)1.0 µl
Final volume: 20 µl
* replace with DEPC-H20 if already included in the 10x RNA amplification buffer
53. Mix gently; incubate at 37 °C for 4 hours.
Prehybridize blots (go to step 58) and prepare a formaldehyde agarose gel (step 57a)
during this incubation.
54. Centrifuge briefly to bring down condensation.
55. Remove 0.5µl and spot onto 1MM Whatman paper (= “TCA-after”).
56. TCA precipitation
a. Wash “TCA-before” and “TCA-after” samples in 10% TCA for 5 min on a rotating platform. Allow a sufficient volume of TCA for the samples to flow freely.
b. Replace with fresh 10% TCA and wash for 5 min. Wash once more for 20 min.
c. Air dry before measuring radioactivity levels, either by counting in a scintillation counter or by listening with a Geiger counter.
There should be a significant increase in the amount of radioactivity measured in the “TCA-after” samples relative to the “TCA-before” samples, signifying successful aRNA synthesis as measured by incorporation of 32P-CTP. You should be able to detect an audible difference using a Geiger counter.
57. Save 2µl of labelled aRNA for gel analysis. Keep on ice until after the remaining probe has been added to blots for hybridization (go to steps 62-67) and the gel is ready.
a. Prepare a formaldehyde agarose gel (preferably before reaction is complete)-
1 liter of 10x MOPS, pH 7: 41.86 g MOPS (= 0.2M final)
6.8 g sodium acetate (= 50 mM final)
20 ml of 0.5 M Na- EDTA pH 8
for 2 mini-gels (60 ml):0.66 g agarose
6 ml 10x MOPS
42.3 ml DEPC-dH2O
- microwave to dissolve agarose; let cool to approx. 55°- 60°C.
- add 11.7 ml formaldehyde in the fume hood
- swirl to mix, without adding bubbles; pour into gelcast(s).
b. Add to aRNA samples: ( 2.0 µl )
formamide 5.0 µl
formaldehyde1.75 µl
10x MOPS 1.0 µl
DEPC-dH2O 0.5 µl
Heat at 80 °C for 5 min to denature.
Cool quickly on ice; centrifuge briefly to bring down condensation.
Add 3 µl of RNA loading dye to samples, then load gel.
Run gels in 1x MOPS buffer at 100 volts for about 1.5 hours.
c. Wash gels in 10% TCA for 30 minutes on a rotating platform.
Replace with fresh 10% TCA and repeat wash for a total of three washes.
d. Flatten gel(s) overnight under paper towels and a heavy weight. Use Saran Wrap underneath the gel and on top of the paper towels to minimize radioactive contamination.
e. Wrap flattened gel(s) in Saran Wrap and expose to X-ray film or phosphorimaging screen.
Gel electrophoresis of the labelled aRNA is highly recommended to assess the size and quality of the labelled aRNA population.
IV. Reverse Northern Blotting
A. Prehybridization
58. Prehybridization solution:
final concentration:
ultrapure formamide50 ml 50%
20x SSC20 ml 4x
50% dextran sulfate20 ml10%
50x Denhardt’s 10 ml 5x
salmon sperm DNA 1 ml 100 µg/ml
Total: 101ml
59. If possible, place blot(s) into a 15 ml or 50 ml conical tube (cDNA side toward the lumen of the tube). Allow sufficient room to avoid overlapping membrane (unless using nylon mesh), while minimizing the volume of prehyb solution needed.
Using a minimal volume will result in a higher concentration of probe and better hybridization.
60. Add prehyb solution to blots. 4 ml per 15 ml tube or 8 ml per 50 ml tube is sufficient. Thoroughly wet the membranes and remove large bubbles between the membrane(s) and the side of the tube.
61. Prehybridize for at least 3 hours at 42 °C in the hybridization oven.
When using 50 ml conicals, it is recommended that the tubes be sealed with parafilm to avoid leakage.
B. Hybridization
62. Heat aRNA probe at 90- 95° C for 5 min.
63. Cool quickly on ice; centrifuge briefly to bring down condensation.
64. Keep all samples on ice to minimize renaturation.
65. Add the probe to the prehyb solution in the tube containing the blot(s). Do not let the probe come in direct contact with the blot.
66. Recap the tube (and re-parafilm); mix well before returning to 42° C oven.
67. Hybridize for at least 16 hours when using dextran sulfate (otherwise 2 days).
C. Washing
68. After hybridization, remove blots directly into a large Tupperware container with 500- 800 ml of 2x SSC, 0.1% SDS. Place on a rotation platform to wash for 30 minutes at room temp.
69. Remove wash. Do a second wash in 2x SSC, 0.1% SDS for 30 min.
70. Remove second wash. Add 0.2x SSC, 0.1% SDS and wash for at least one hour.
D. Autoradiography
71. Remove blots from third wash solution and place directly into plastic sealable bags. Keep the blots moist in case more washing is required, or if you desire to strip the blots and reuse them.
72. Seal the plastic bag. Expose the blots to X-ray film or phosphorimaging screen.
APPENDIX:
A. Amplification Buffers (for 50 ml in DEPC-H2O):
10x RT
500mM Tris-base, pH 8.3 3.03g (pH with HCl)
1.2M KCl4.47g
100mM MgCl21.02gMix. Filter through 0.2µm filter.
Store in 1ml aliquots at -20 °C.
10x 2nd Strand
1M Tris-base, pH 7.46.06g (pH with HCl)
200mM KCl0.746g
100mM MgCl21.02g
400mM (NH4)2SO42.64g Mix. Filter through 0.2µm filter.
Store in 1ml aliquots at -20 °C.
50mM DTTadd later*
10x RNA Amplification
400mM Tris-base, pH 7.52.42g (pH with HCl)
70mM MgCl20.712g
100mM NaCl0.292g Mix. Filter through 0.2µm filter.
Store in 1ml aliquots at -20 °C.
20mM spermidine add later*
10x KFI
200mM Tris-base, pH 7.51.21g (pH with HCl)
100mM MgCl21.02g
50mM NaCl0.146g Mix. Filter through 0.2µm filter.
Store in 1ml aliquots at -20 °C.
50mM DTTadd later*
*The buffer can be stored longer (for years) without this component.
B. Special reagents
T7-oligo(dT)24 oligonucleotide-linked magnetic beads: please refer to separate protocol.
T7-oligo(dT)24 oligonucleotide:
5’-AAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGCGC(T)24
AMV-RT (avian myeloblastosis virus reverse transcriptase):
25 units/ µl Seikagaku America (800-237-4512) catalog #1-120248-2 (5000u)
T7 RNA polymerase (cloned) :
1000 units/µlEpicentre Technologies (800-284-8474)catalog# TH950K (50,000u)
C. Preparation of cDNA blot
1. Linearize cDNAs of interest using an appropriate restriction enzyme. Check for complete digestion before proceeding. There is no need to ethanol-precipitate the cDNAs. The integrity of linearized plasmid that has been stored for more than a couple weeks should be rechecked on a gel before proceeding.
Remember to include linearized plasmid vector as a background control.
2. DEPC- treat the top slotted portion of the blotting apparatus (Milliblot-S System).
3. Cut 3 pieces of 3MM Whatman paper and one piece of nitrocellulose or nylon membrane to fit within the rubber gaskets of the apparatus.
4. Using clean gloves or DEPC-treated forceps, wet the Whatman papers and membrane in 10x SSC.
5. Assemble the apparatus from the bottom. Place the three sheets of Whatman paper on top of the middle slotted piece, then the membrane. Make sure that the paper and membrane stay within the gaskets to ensure a tight seal. Roll out any air bubbles that may have formed between the layers before placing the top slotted piece onto the apparatus and completing the assembly. Add 10x SSC to the wells to moisten and set aside.
If using vacuum application, check at this time to ensure that suction is gentle enough
(ie. at least 5 min for 100µl volume) before loading samples.
6. Prepare 0.5 µg of linearized cDNA in 100µl 10x SSC per well.
7. a. Heat denature samples at 85-90°C for 5 minutes.
b. Cool quickly on ice.
c. Centrifuge briefly to bring down condensation.
d. Keep the samples on ice while loading.
8. Remove excess 10x SSC from the wells of the blotting apparatus (Shake vigorously into the sink). Add 100 µl sample to each well.
It is a good idea to carefully write out the layout of cDNAs before loading and to note any deviations immediately after loading.
9. Apply a gentle vacuum to draw samples through the slots. This should take at least 5 minutes or you risk pulling the samples through the membrane. Alternatively, one can use gravity to draw the samples through (although this will take several hours).
10. Once samples have been drawn through and the wells are completely dry, disassemble the apparatus. Take note of the orientation of the blot (perhaps cut a corner) before removing the membrane and placing it on Whatman paper to dry.
10. UV-crosslink the cDNA to the membrane using the Stratalinker.
D. Genome Systems protocol for stripping blots
1. Place filters in hyb bottle with DNA side to the lumen.
2. Add 50 ml of 0.4M NaOH for 45 min at 45 °C and 20 rpm.
3. Rinse once with 50 ml of 0.2M Tris-HCl, pH 7.2, 0.1% SDS, 0.1x SSC.
4. Wash two times in 100 ml of 0.2M Tris-HCl, pH 7.2, 0.1% SDS, 0.1x SSC for 10 min at room temp. and 20 rpm
5. Reimage moist filters to ensure stripping was complete.
6. If stripping was not complete, repeat process with 100 ml of 0.4M NaOH prewarmed to 50 °C for 1 hour at 50 °C and 20 rpm.
7. Repeat washes as above.
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