PROTOCOL FOR SINGLE CELL mRNA AMPLIFICATION by tyndale

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									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              3ul
               1x RT buffer                                    17.5ul
               dGTP (10mM)                                        1ul
               dATP (10mM)                                        1ul
               dTTP (10mM)                                        1ul
               dCTP (10mM)                                        1ul
               Oligo-dT-T7(100ng/ul)                              1ul
               DTT(100mM)                                         3ul
               RNasin                                  0.5ul
               AMV-RT(25U/ul)                                    1ul


               total volume                                     30ul

2.Mix gently; incubate at 42C for 90min.
3.Phenol/chloroform extraction cDNA:

               DD-H2O                                           105ul
               3M Na acetate                                     15ul
               chloroform                                        75ul
               buffer-saturated phenol                           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                                      5ul
       100mMDTT                                                   2ul
       4 dNTPs(2.5mM each)                                        5ul
       random hexamers (100ng/ul)                                 1ul
        T4 DNA polymerase (5U/ul)                               0.2ul(1U final)
       Klenow (5U/ul)                                           0.5ul(2U final)
       DDH2O                                                   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                                                 21ul
       10x KFI buffer                                          3ul
       100mM DTT*                                              3ul
       4 dNTPs                                                 3ul
       T4 DNA polymerase(5U/ul)                              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                                                 85ul
       3M sodium acetate                                      15ul
       chloroform                                             75ul
       buffer-saturated phenol                                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                                 2.0ul
        10x TSC buffer(RNA amplification buffer)        2.5ul
        20mM spermidine*                                2.0ul
        4rNTPs(with UTP)(2.5mM each)                    2.5ul
        100mM DTT                                        1ul
        RNasin                                0.5ul
        T7 RNA polymerase (1000U/ul)                       1ul (1000U final)
        DD-H2O                                          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                                          95ul
       3M ammonium acetate*                            15ul
       chloroform                                      75ul
       buffer-saturated phenol                         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:                     (19.5ul)

       5x first strand buffer                        3.0ul
       4 dNTPs (2.5mM each)                          3.0ul
       random hexamer (100ng/ul)                      1.0ul
       100mM DTT                                      2.0ul
       RNasin                                0.5ul
       Superscript RT                                1.0ul

       final volume                                  30ul

34.Mix gently; incubate @ 42C for 90 min.

35.Phenol-chloroform extract ss-cDNA. add:

       DD-H2O                                        105ul
       3M sodium acetate                              15ul
       chloroform                                     75ul
       buffer-saturated phenol                        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 DD-
H2O.

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                                 2ul
       100mM DTT*                                     2ul
       4 dNTPs(2.5mM each)                            2ul
       T7-oligo(dT)24 primer(100ng/ul)                1ul
       T4 DNA polymerase (5U/ul)                    0.2ul (1U final)
       Klenow (5U/ul)                                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 self-
hybridization)the amount of aRNA probe available for hybridization to cDNA on the
blot.

53.Add to 2nd strand reaction:                      (20ul)

       DD-H2O                                       21ul
       10x KFI buffer                                3ul
       100mM DTT*                                    3ul
       4 dNTPs(2.5mM each)                           3ul
       T4 DNA polymerase (5u/ul)                   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                                       85ul
       3M sodium acetate                            15ul
       chloroform                                   75ul
       buffer-saturated phenol                      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                      2.0ul
      20mM spermidine*                                  2.0ul
      100mM DTT                                         1.0ul
      3 rNTPs(ATP,GTP,UTP)(2.5mM each)                  2.0ul
      100uM CTP                                         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:
                                                                      final concentration
                      ultrapure formamide                 50ml              50%
                      20x SSC                             20ml               4x
                      50% dextran sulfate                 20ml               10%
                      50x Denhardt’s                      10ml               5x
                      salmon sperm DNA                     1ml            100ug/ml

                                    Total                 101ml

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                        3.03g (pH with HCl)
   1.2M KCl                                       4.47g
   100mM MgCl2                                    1.02g       Mix. Filter through 0.2µm
   filter. Store in 1ml aliquots at -20 °C.

10x 2nd Strand

   1M Tris-base, pH 7.4                           6.06g (pH with HCl)
   200mM KCl                                      0.746g
   100mM MgCl2                                    1.02g
   400mM (NH4)2SO4                                2.64g       Mix. Filter through 0.2µm
   filter. Store in 1ml aliquots at -20 °C.
   50mM DTT                                       add later*

10x RNA Amplification

   400mM Tris-base, pH 7.5                        2.42g (pH with HCl)
   70mM MgCl2                                     0.712g
   100mM NaCl                                     0.292g      Mix. Filter through 0.2µm
   filter. Store in 1ml aliquots at -20 °C.
   20mM spermidine                                         add later*

10xKFI

   200mM Tris-base, pH 7.5                        1.21g (pH with HCl)
   100mM MgCl2                                    1.02g
   50mM NaCl                                      0.146g      Mix. Filter through 0.2µm
   filter. Store in 1ml aliquots at -20 °C.
   50mM DTT                                       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.   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.

   11. 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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