NO Beads
Revised version 8-27-99
PROTOCOL FOR SINGLE CELL mRNA AMPLIFICATION/
REVERSE NORTHERN ANALYSIS
I. First round amplification
A.First strand cDNA synthesis
1.Add to single cell:
Cell contents plus recording solution
1x RT buffer
dGTP (10mM)
dATP (10mM)
dTTP (10mM)
dCTP (10mM)
Oligo-dT-T7(100ng/ul)
DTT(100mM)
RNasin
AMV-RT(25U/ul)
3ul
17.5ul
1ul
1ul
1ul
1ul
1ul
3ul
0.5ul
total volume
1ul
30ul
2.Mix gently; incubate at 42C for 90min.
3.Phenol/chloroform extraction cDNA:
DD-H2O
3M Na acetate
chloroform
buffer-saturated phenol
105ul
15ul
75ul
75ul
final volume
300ul
4.Vortex for 10 sec. Centrifuge for 3 min. Carefully remove~145ul aqueous (top)
phase to a new tube.
5.Precipitate with ethanol. Add 300ul 100% ice-cold ethanol,1ul tRNA(5ug)
6.Leave on dry ice for least 20-30 mins. . Centrifuge at 18,000rpm at 4C for 30 min.
B.Second strand cDNA synthesis
7. Dry & resuspend pellet in 20ul DDH2O.
8.Heat at 90-95C for 3 min to denature RNA:DNA hybrid.
9. Cool quickly on ice; centrifuge briefly to bring down condensation.
10.Add to sample:
(20ul)
10x 2nd strand buffer
100mMDTT
4 dNTPs(2.5mM each)
random hexamers (100ng/ul)
T4 DNA polymerase (5U/ul)
Klenow (5U/ul)
DDH2O
5ul
2ul
5ul
1ul
0.2ul(1U final)
0.5ul(2U final)
16.3ul
final volume
50ul
11.Mix gently; incubate at 14C for a minimum of 5 hrs, or overnight.
C.Blunt-end treatment
12. S1 nuclease cut or blunt –end repair (if go into reverse northern):
dilute 1ul of S1 nuclease in 400ul of 10x S1 buffer.
13.Add to 2nd strand:
DDH2O
400ul
10x S1 buffer
50ul
diluted S1 nuclease (1U)
1ul
tRNA
1ul
14. Incubate at 37C for 5 min.
15. Extract with phenol/chloroform(0.5x):
add to blunt-end treated 2nd strand sample
452ul
chloroform
226ul
buffer-saturated phenol
226ul
16. Vortex 10 sec. centrifuge for 3min. extract top aqueous layer
into a new tube.
17.Precipitate with 1ml 100% ETOH on dry ice for 20-30 mins.
18.Centrifuge 18,000rpm @4C for 30 min., dry pellet.
19.Resuspend pellet in 20ul of DDH2O, add
DD-H2O
10x KFI buffer
100mM DTT*
4 dNTPs
T4 DNA polymerase(5U/ul)
21ul
3ul
3ul
3ul
0.2ul
final volume
50ul
*replace with DD-H2O if already included in the 10xKFI buffer
20.Incubate at 37C for 15-30 min.
21. Phenol-chloroform extract ds-cDNA. Add:
DD-H2O
3M sodium acetate
chloroform
buffer-saturated phenol
85ul
15ul
75ul
75ul
final volume
300ul
22.vortex for 3 sec. Centrifuge for 3 mins. Carefully transfer ~145ul top aqueous layer to
a new tube.
23.Add 300ul 100% ice-cold ETOH on dry ice for 20-30mins.Centrifuge at 18,000rpm at
4C for 30 mins.
24.Resuspend pellet in 20ul of DD-H2O. Drop dialyze 10-20ul sample against 50 ml
DD-H2O for at least 4 hrs.
D.First round aRNA amplification (cold reaction)
25. use 1/10 of total sample & add:
Dialyzed sample
10x TSC buffer(RNA amplification buffer)
20mM spermidine*
4rNTPs(with UTP)(2.5mM each)
100mM DTT
RNasin
0.5ul
T7 RNA polymerase (1000U/ul)
DD-H2O
2.0ul
2.5ul
2.0ul
2.5ul
1ul
1ul (1000U final)
8.5ul
final volume
20ul
*replace with DD-H2O if already included in the 10x RNA amplification buffer.
26. Mix gently; incubate @ 37C for 4 hrs.
27.Phenol/chloroform extract aRNA. Add to tube :
DD-H2O
3M ammonium acetate*
chloroform
buffer-saturated phenol
95ul
15ul
75ul
75ul
final volume
300ul
*ammonium acetate if more efficient than sodium acetate at removing free NTPs. Adding
salt at this step(instead of subsequent ethanol step) improves separation of the aqueous
and organic phases.
28. Vortex for 10 sec. Centrifuge for 3 min. Carefully remove 145ul aqueous (top) layer
to a new tube.
29. Add
tRNA* (1ug/ul)
1ul
100% ice-cold ethanol
450ul
*use of a carrier is highly recommended to aid precipitation of miniscule amounts of
RNA (especially from single cells.) However, tRNA can sometimes cause artefacts in
PCR reactions. While this is usually not a problem wtih routine PCR analysis, it may be
critical when doing differential display-PCR, for example. Glycogen is also thought to
interfere with many manipulaitons. Other carriers have not been tested, but may be
appropriate in these cases.
30.Precipitate on dry ice for 20-30mins. Centrifuge 18,000 @ 4C for 30 mins.
Resuspend the pellet in 19.5ul of DD-H2O.
II. Conversion of aRNA to double-stranded cDNA
A. First strand cDNA synthesis
31. Denature 19.5ul aRNA sample at 90-95 C for 3 min.
32.Quick cool, quick spin to bring down condensation.
33.Add to denatured aRNA sample:
5x first strand buffer
4 dNTPs (2.5mM each)
random hexamer (100ng/ul)
100mM DTT
RNasin
Superscript RT
final volume
(19.5ul)
3.0ul
3.0ul
1.0ul
2.0ul
0.5ul
1.0ul
30ul
34.Mix gently; incubate @ 42C for 90 min.
35.Phenol-chloroform extract ss-cDNA. add:
DD-H2O
3M sodium acetate
chloroform
buffer-saturated phenol
105ul
15ul
75ul
75ul
final volume:
300ul
36.Vortex for 10 sec. Centrifuge for 3 min. Carefully transfer 145ul aqueous (top) layer
to a new tube.
37.Add 300 ul 100% ice-cold ethanol to precipitate on dry ice for 20-30 mins.
38. Centrifuge at 18,000rpm at 4C for 30 min. Resuspend the pellet in 12.3ul of DDH2O.
B. Second strand cDNA synthesis
39.Heat at 90-95 C for 3 min to denature aRNA:DNA hybrid.
41. Quick cool, quick spin to bring down condensation.
42.Add to sample:
10x KFI buffer
100mM DTT*
4 dNTPs(2.5mM each)
T7-oligo(dT)24 primer(100ng/ul)
T4 DNA polymerase (5U/ul)
Klenow (5U/ul)
2ul
2ul
2ul
1ul
0.2ul (1U final)
0.5ul (2U final)
final volume:
20ul
*replace with DD-H2O if already included in the 10x KFI buffer
43. Mix gently; incubate at 14C overnight.
44.Centrifuge briefly to bring down condensation.
C. Blunt-end reaction
45. S1 nuclease cut or blunt –end repair (if go into reverse northern):
dilute 1ul of S1 nuclease in 400ul of 10x S1 buffer.
46.Add to 2nd strand:
DDH2O
400ul
10x S1 buffer
50ul
diluted S1 nuclease (1U)
1ul
tRNA
1ul
47. Incubate at 37C for 5 min.
48. Extract with phenol/chloroform(0.5x):
add to blunt-end treated 2nd strand sample
452ul
chloroform
226ul
buffer-saturated phenol
226ul
49. Vortex 10 sec. centrifuge for 3min. extract top aqueous layer
into a new tube.
50.Precipitate with 1ml 100% ETOH on dry ice for 20-30 mins.
51.Centrifuge 18,000rpm @4C for 30 min., dry pellet.
52.Resuspend pellet in 20ul of DDH2O, add
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 selfhybridization)the amount of aRNA probe available for hybridization to cDNA on the
blot.
53.Add to 2nd strand reaction:
(20ul)
DD-H2O
10x KFI buffer
100mM DTT*
4 dNTPs(2.5mM each)
T4 DNA polymerase (5u/ul)
21ul
3ul
3ul
3ul
0.2ul
final volume
50ul
*replace with DDH2O if already included in the 10x KFI buffer
54. Incubate at 37C for 15-30min.
55. Phenol-chloroform extract ds-cDNA. add
DD-H2O
3M sodium acetate
chloroform
buffer-saturated phenol
85ul
15ul
75ul
75ul
final volume:
300ul
56. Vortex for 10 sec.Centrifuge for 3min. Carefully transfer 145ul (top) aqueous phase
to a new tube.
57.Add 300ul 100% ice-cold ethanol to precipitate on dry ice for 20-30mins.
58. Centrifuge @18,000rpm at 4C for 30 min. Resuspend the pellet in 20ul of DD-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 10-20ul sample against 50ml DD-H2O for at least 4 hrs.
52. Combine:
1/10 dialyzed sample plus DD-H2O
7.7 ul
10x RNA amplification buffer
20mM spermidine*
100mM DTT
3 rNTPs(ATP,GTP,UTP)(2.5mM each)
100uM CTP
2.0ul
2.0ul
1.0ul
2.0ul
0.8ul
RNasin
0.5ul
alpha-[32P}-CTP(3000Ci/mmol;1mci/100ul
3.0ul(4mM final=80pmol)
-remove 0.5ul and spot onto 1MM whatman paper(=TCA before)
add T7 RNA polymerase (1000U/ul)
1.0ul
final volume
20ul
*replace with DD-H2O if already included in the 10x RNA amplification buffer
53.Mix gently; incubate at 37C for 4-8 hrs. (6 hrs. is recommended)
Prehybridize blots (go to step 57 ).
54. Centrifuge briefly to bring down condensation.
55.Remove 0.5ul & spot onto 1MM Whatman paper (=TCA after)
56.TCA precipitation
a.Wash “TCA-before” & “TCA-after” samples in 10% TCA for 5 min on a
rotating plateform. 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 a RNA
synthesis as measured by incorporation of 32P-CTP. You should be able to detect an
audible difference using a Geiger counter.
IV. Reverse Northern Blotting
A. Prehybridization
57.Prehybridization solution:
ultrapure formamide
20x SSC
50% dextran sulfate
50x Denhardt’s
salmon sperm DNA
50ml
20ml
20ml
10ml
1ml
Total
101ml
final concentration
50%
4x
10%
5x
100ug/ml
58.Place blot(s) into a 15ml or 50ml 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.
59.Add prehyb solution to blots. 4ml per 15ml tube or 8 ml per 50ml tube is
sufficient. Thoroughly wet the membranes and remove large bubbles between
the membrane(s) and the side of the tube.
60. Prehybridize for at least 3 hrs at 42C in the hybridization oven.
When using 50ml conicals, it is recommended that the tubes be sealed with
parafilm to avoid leakage.
B.Hybridization
61.Heat aRNA probe at 90-95C for 5 min.
62.Cool quickly on ice;centrifuge briefly to bring down condensation.
63.Keep all samples on ice to minimize renaturation.
64.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.
65.Recap the tube(and re-parafilm); mix well before returning to 42C oven.
66.Hybridize for at least 16 hrs. when using dextran sulfate (otherwise 2 days)
C.Washing
67.After hybridization, remove blots directly into a large Tupperware container with
500-800ml of 2x SSC, 0.1% SDS. Place on a rotation platform to wash for 30
mins. at room temp.
68. Remove wash. Do a second wash in 2x SSC, 0.1% SDS for 30 min.
69.Remove second wash. Add 0.2x SSC, 0.1%SDS & wash for at least one hour.
D.Autoradiography
70. 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 & reuse them.
71. 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
1.2M KCl
100mM MgCl2
filter. Store in 1ml aliquots at -20 °C.
3.03g (pH with HCl)
4.47g
1.02g
Mix. Filter through 0.2µm
10x 2nd Strand
1M Tris-base, pH 7.4
200mM KCl
100mM MgCl2
400mM (NH4)2SO4
filter. Store in 1ml aliquots at -20 °C.
50mM DTT
6.06g (pH with HCl)
0.746g
1.02g
2.64g
Mix. Filter through 0.2µm
add later*
10x RNA Amplification
400mM Tris-base, pH 7.5
70mM MgCl2
100mM NaCl
filter. Store in 1ml aliquots at -20 °C.
20mM spermidine
2.42g (pH with HCl)
0.712g
0.292g
Mix. Filter through 0.2µm
add later*
10xKFI
200mM Tris-base, pH 7.5
100mM MgCl2
50mM NaCl
filter. Store in 1ml aliquots at -20 °C.
50mM DTT
1.21g (pH with HCl)
1.02g
0.146g
Mix. Filter through 0.2µm
add later*
*The buffer can be stored longer (for years) without this component.
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.
b.
c.
d.
Heat denature samples at 85-90°C for 5 minutes.
Cool quickly on ice.
Centrifuge briefly to bring down condensation.
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.
11. UV-crosslink the cDNA to the membrane using the Stratalinker.
D. Genome Systems protocol for stripping blots
1.
2.
3.
4.
Place filters in hyb bottle with DNA side to the lumen.
Add 50 ml of 0.4M NaOH for 45 min at 45 °C and 20 rpm.
Rinse once with 50 ml of 0.2M Tris-HCl, pH 7.2, 0.1% SDS, 0.1x SSC.
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.