Esophageal Adenocarcinoma Methods and Protocols

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11. Standard thermal cycler.


12. Centrifuge with plate holders.


13. Microcentrifuge.


14. Vortex.


15. Polypropylene tube.


16. Reagent tubes with caps.


17. 96-Well optical reaction plates.


18. Optical adhesive covers.


19. Primers for target miRNAs.


20. Internal control primer.

3 Methods

1. Excise the tissue samples from patients or healthy control or
   take it from storage (see Note 1) or cells from culture.


2. Add 700 μL lysis buffer (Triazole) onto the specimen or cells
   and homogenize the samples.


3. Incubate the samples on benchtop at room temperature for
   5 min.


4. Add 140 μL chloroform to the tubes containing the homoge-
   nate and cap securely. Shake the tubes vigorously for mixing
   the component for 15 s.


5. Keep the tubes on benchtop for 2–3 min.


6. Centrifuge the tubes for 15 min at 12,000 × g at 4 °C (see
   Note 2).


7. Take the aqueous phase into new 1.5 μL tubes and add 1.5
   volumes (usually 525 μL) of 100% ethanol. Mix thoroughly by
   pipetting up and down several times.


8. Place 700 μL of the samples to the isolating column, close the
   lid, and centrifuge at 8000 × g for 15 s at room temperature.


9. Repeat step 8 with remaining samples.


10. Add 700 μL wash buffer to the column and centrifuge at
    8000 × g for 15 s at room temperature (repeat washing couple
    of times).


11. Transfer column into new 1.5 μL collection tubes and elute
    with 30–50 μL RNase-free water by centrifuging 1 min at
    8000 × g at room temperature.

The principle of microarrays is based on the Watson–Crick base-

pairing nature of nucleic acids. Thousands of probes can be spot-

ted on slides and the brightness of individual spots can be used to

3.1 Extraction of
RNA Including miRNAs
from Tissue, Blood, or
Cells

3.2 Microarrays

MicroRNA Detection and Esophageal Adenocarcinoma