Esophageal Adenocarcinoma Methods and Protocols

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  1. Denature by incubating at 95 °C for 2 min and immediately
    cooling down on ice (see Note 8).

  2. Put the backing slide in the chamber base.

  3. Add 400 μL of the sample mixture onto the gasket slide (see
    Note 9).

  4. Place a microarray slide on top of the backing gasket slides
    with the array side facing the target samples (see Note 10).

  5. Clamp the array/backing slide into the hybridization
    chambers.

  6. Incubate at 56 °C for 16 h in a hybridization oven with
    rotation.

  7. Prepare the first wash buffer containing 60 mL 20× salt buffer,
    12 mL 10% detergent solution, and 528 mL nuclease-free
    water and pre-warm to 56 °C overnight.

  8. Remove array/backing slide sandwich from hybridization
    chamber, separate the slides from the backing slide using plas-
    tic forceps, and place slides in a submerged slide rack inside a
    jar containing the first wash buffer.

  9. Wash the slide for 2 min in the first wash buffer at 56 °C.

  10. Rinse the slides with the second wash buffer at room tempera-
    ture containing 20 mL 20× salt buffer and 380 mL nuclease-
    free water.

  11. Wash the slide for 2 min in the second wash buffer and at room
    temperature in a new jar.

  12. Wash the slide for 2 min in the third wash buffer and at room
    temperature in a new jar containing 2 mL 20× salt buffer and
    198 mL nuclease-free water.

  13. Remove the slides gently allowing the buffer to run off.

  14. Transfer the slide rack to a new staining dish with 99% ethanol
    at room temperature for a few seconds.

  15. Remove the slides gently allowing the wash to run off.

  16. Quick-dry the slides by centrifuging at 600 × g for 2–5 min.

  17. Scan slides immediately after drying.

  18. Analyze images using appropriate software.


Quantitative reverse transcriptase polymerase chain reaction (qRT-
PCR) has become one of the most widely used techniques for
detection and comparison of RNA levels due to the simplicity,
specificity, and sensitivity that it offers [ 35 ]. In RT-qPCR, the first
step is to extract miRNA and convert it to cDNA by reverse tran-
scription. As the length of a miRNA is very small and in fact similar
to DNA primer, cDNA synthesis from miRNAs can be challeng-
ing. To overcome this, the most common approaches include

3.3 Quantitative
Real-Time Polymerase
Chain Reaction
(RT-qPCR)


MicroRNA Detection and Esophageal Adenocarcinoma
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