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  1. Comparible RNAs were isolated from different treatments using my favorite method or any other methods you like, and dissolved in RNA water.
  2. DNase treatment of 20ug total RNA, 37oC, 30' with the following reaction mix:
  3. Extract with phenol/chloroform
  4. Precipate with 1/10Vol 3M NaAc, pH4.8 and 2.5Vol ethanol, -70oC, 20min.
  5. Spin at 4oC, 5min.
  6. Resuspend in 20ul RNA water
  7. Denature the RNA at 650C, 5min., then ice.
  8. Dilute Oligo-dTplus (T12NA, T12NT, T12NG, T12NC) primers to final concentration 50uM.
  9. Reverse transcription (in 4 tubes):
  10. Make PCR reaction stock (total volume 100ul):
  11. Mix different RNA samples and 4 different oligo-dTplus primers (4 tubes for each RNA samples) for T12NA, T12NT, T12NG or T12NC repectively:
  12. Divide the solution above in each tube to 3 small Eppendorf tubes,19ul each, add 1ul of each random primer (10mM stock) to each tube (in my case, three different random primers were used), and one drop of mineral oil to each tube.
  13. PCR cyclings
  14. Prepare 6% sequencing gel:
  15. Add 5ul loading buffer (same as used in sequencing gel) to each PCR reaction tube.
  16. Denature the reaction at 95oC, 3min.
  17. Prerun the gel at 1300V for 20min. in 1XTBE buffer.
  18. Load the samples, paralell between corresponding samples.
  19. Run the gel at 1300V for 4-5Hrs, 1100V in gradient gel.
  20. Fix the gel with 10% ethanol for 15min.
  21. Dry the gel in a gel-drier for 1hr.
  22. Exposure to X-ray film overnight.
  23. Cut the differential bands from the gel, put in 50ul H2O, 37oC, 1hrs, then boil for 5min.
  24. Add 1/10 vol. of 3M NaAc, and 150ul 100% ethanol, -70oC o/n.
  25. Spin at 4oC 5min.
  26. Suck out the ethanol, spin dry. Then disolve the pellet in 10ul TE buffer.
  27. Secondary PCR
  28. This step can also be carried out by using a PCR stock solution to reduce the pepetting. To make the PCR stock, mix:
    • 840ul pure water
    • 54ul 2mM dNTP
    • 140ul 10XPCR buffer
    • 280ul 50% glycerol
    • For a 50ul reaction, use:
    • 46.5ul the stock
    • 1ul DNA fragment
    • 1ul 3'primer
    • 1ul 5' primer
  29. PCR cycling, same as before(#13), 0.5ul Taq polymerase was added after hot-start
  30. Run the samples with 1.5% agarose gel in 1/2TBE buffer
  31. Note: To have a pure PCR product, it will help to run the secondary PCR product together with the original in a sequencing gel and cut out the band again.
  32. Northern blot and probe with 32P-dATP labeled bands to confirm the difference.

  33. Comments or Questions? Send to me

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