Esophageal Adenocarcinoma Methods and Protocols

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  1. Mix and centrifuge.

  2. Dispense 1.0 μL (or alternative according to the kit manual) of
    20× MicroRNA Assay mix into the corresponding PCR reac-
    tion tube.

  3. Transfer above into corresponding wells.

  4. Transfer 1.33 μL of the reverse transcription product from the
    96-well reverse transcription plate into the 96-well PCR plate
    and seal with optical adhesive cover.

  5. Mix and centrifuge.

  6. Load the PCR plate and run real-time PCR at standard condi-
    tions: one cycle of 10 min at 95 °C for enzyme activation and
    40 cycles of PCR (15 s at 95 °C, followed by 60 s at 60 ° C)
    (see Note 16).

  7. Transfer the results to Excel or an alternative spreadsheet and
    analysis software.


4 Notes



  1. Measure the tissue weight. Do not take more than 50 mg of
    tissues and do not thaw tissues before adding lysis reagents.

  2. After centrifugation, the sample separates into three layers: an
    upper aqueous phase containing RNA including small miR-
    NAs, a white interphase, and a lower red organic phase. The
    volume of the aqueous phase should be approximately 350 μL.

  3. Instead of thawing the enzymes in this step, you should flick
    the enzyme tubes 3–4 times and subsequently spin them down
    using a table microcentrifuge machine.

  4. 3 μL of total RNA should contain approximately between 0.25
    and 1.5 μg. If unsure of the concentration, you should quanti-
    tate your RNA sample using a quantitation kit.

  5. You can leave on ice for a minimum of 2 min and a maximum
    of 15 min.

  6. Make sure the sample is protected from light to not interfere
    with the dye.

  7. If you notice that there is precipitation in the hybridization
    buffer, you can warm the solution at 56 °C and shake the jar to
    dissolve.

  8. Once again, make sure the sample is protected from light. You
    should leave on ice for a minimum of 2 min and maximum of
    15 min.

  9. Start close to one end and slowly dispense your sample while
    you move your pipette to the opposite end so that the sample


MicroRNA Detection and Esophageal Adenocarcinoma
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