Esophageal Adenocarcinoma Methods and Protocols

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  1. RNase-free water.

  2. 100% ethanol.

  3. Centrifuges.

  4. RNase-free, sterile-filtered water.

  5. miRNA isolation kit.

  6. 2× hybridization buffer:100 mM 2-(N-morpholino) ethane-
    sulfonic acid, 1 M [NaCl], 20 mM ethylenediaminetetraacetic
    acid (EDTA), 0.01% Tween-20. Do not autoclave. Store at
    2–8 °C, and protect from light. Discard solution if turned to
    yellow.

  7. 20× salt buffer: 3 M sodium chloride, 300 mM tri-sodium
    citrate. Adjust pH to 7.0 with HCl.

  8. 10% detergent solution.

  9. miRNA microarray slides.

  10. microRNA array labeling kit.

  11. Hybridization chamber kit.

  12. Hybridization gasket slide kit.

  13. Hybridization oven with rotation.

  14. Microarray scanner.

  15. 99% ethanol.

  16. Microcentrifuge.

  17. Vortex.

  18. Pipettes (positive-displacement, air-displacement, or multichan-
    nel): 1–20 μL range, 20–200 μL range, 100–1000 μL range.

  19. Pipette tips, aerosol resistant, nuclease-free: 1–20 μL range,
    20–200 μL ranges, 100–1000 μL range.

  20. RNase-free microcentrifuge tube.

  21. RNase-free, sterile-filtered water.

  22. MiRNA isolation kit.

  23. 20× miRNA Assay Mix.

  24. 10× reverse transcription buffer: 0.75 M KCl, 0.1 M MgCl 2 ,
    0.5 M Tris–HCl. Adjust pH to 8.6 at 25 °C.

  25. dNTP mix w/dTTP (100 mM total).

  26. RNase Inhibitor (20 U/μL).

  27. Reverse transcriptase.

  28. miRNA reverse transcription primer.

  29. Universal PCR Master Mix.

  30. Real-time thermal cycler.


2.2 Microarrays


2.3 RT-qPCR


Moein Amin et al.
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