Targeted Differential Display

 

This method was successful in our lab using prostate tissue and for our specific objectives. Investigators must be aware that they will need to tailor the following protocol for their own research objectives and tissue under study.

The protocol below is one example of differential gene expression analysis of cells obtained from microdissected tissue.

1. Materials

1. 1. Stratagene RAP-PCR kit
   2. 5X RT buffer (GenHunter)
   3. dNTP, 250 µM (GenHunter)
   4. dNTP, 25 µM (GenHunter)
   5. RNase inhibitor (Perkin Elmer)
   6. Random hexamer primers (Perkin Elmer)
   7. MMLV reverse transcriptase, 100 units/µl (GenHunter)
   8. 10X PCR buffer (Perkin Elmer)
   9. AmpliTaq DNA polymerase (Perkin Elmer)
   10. DEPC- treated H ₂ O
   11. 3 M Na acetate, pH 5.2 (Life Technologies)
   12. 100% ethanol
   13. 2X loading dye (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol )

2. Methods (Under sterile, RNase free conditions)

Note: The protocol was utilized to screen for tumor-specific alterations in transcript levels using a variety of screening approaches and primer combinations, including the RAP-PCR arbitrary primers from Stratagene and degenerate primers directed against known protein motif DNA sequences. Exact conditions must be determined for each differential display study depending on the number of cells utilized, the primer sets, and the number of transcripts the investigator wishes to screen.

A: Microdissection and RNA Isolation

1. 1. Dissect approximately 5000-10,000 cells from frozen sections cut to a thickness of 12 µm. Complete the microdissection within 30 minutes of preparation of the tissue sections.
   2. Isolate total RNA, as from protocol in RNA-based Studies of Microdissected Tissues.

TIP: Only use cells from frozen tissue that is extremely well-preserved.

TIP: If anchored, oligo-dT-based differential display is to be performed (e.g., with the GenHunter kit), it is recommended to start with RNA recovered from a substantially larger number of microdissected cells.

B: Reverse Transcription

1. 1. Add 24 µl H ₂ O and 1 µl RNase inhibitor, 20 U/µl, to RNA pellet.
   2. Resuspend the RNA pellet with gentle tapping.
   3. Quick spin.
   4. Aliquot 12 µl into 2 tubes for the (+) and (-) RT reactions.
   5. Prepare additional control tubes using the following in place of the sample RNA:
      • A positive control prepared with known, high-quality RNA
      • A negative control prepared from either a LCM cap containing no tissue or RNase-free water
   6. Prepare sufficient volume of the following reaction mixture for each tube:

       

      Reaction mixture/tube   4 µl 5X RT buffer 2 µl dNTP mix (250 µM) 1 µl Random hexamer primers   Total volume = 7 µl