PROTOCOL FOR SINGLE CELL mRNA AMPLIFICATION

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					 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-H2O                            24 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-H2O                         10 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 ddH 2O (0.1% final concentration).

       11. Mix gently; incubate at 42 °C for 60-90 min.
                                               1
            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-H2O                                                         28.3 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-H2O                                                         11.5 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


                                                     2
   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.




                                                   3
      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 ethanol               450 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

                                                      4
          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

                                                5
             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-H2O                  7.7 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 CTP                                                      0.8 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.
                                                     6
           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.




                                                  7
   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
                            formaldehyde               1.75 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.




                                                  8
IV. Reverse Northern Blotting

   A. Prehybridization

      58. Prehybridization solution:
                                                                             final concentration:
                       ultrapure formamide                 50 ml                     50%
                       20x SSC                             20 ml                      4x
                       50% dextran sulfate                 20 ml                     10%
                       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.

                                                      9
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.




                                           10
                                        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*


10x KFI

   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.


B. Special reagents

T7-oligo(dT)24 oligonucleotide-linked magnetic beads: please refer to separate protocol.
                                               11
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/µl   Epicentre Technologies (800-284-8474)     catalog# TH950K (50,000u)




                                             12
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.

                                                     13
   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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