261
- Standard thermal cycler.
- Centrifuge with plate holders.
- Microcentrifuge.
- Vortex.
- Polypropylene tube.
- Reagent tubes with caps.
- 96-Well optical reaction plates.
- Optical adhesive covers.
- Primers for target miRNAs.
- Internal control primer.
3 Methods
- Excise the tissue samples from patients or healthy control or
take it from storage (see Note 1) or cells from culture. - Add 700 μL lysis buffer (Triazole) onto the specimen or cells
and homogenize the samples. - Incubate the samples on benchtop at room temperature for
5 min. - 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. - Keep the tubes on benchtop for 2–3 min.
- Centrifuge the tubes for 15 min at 12,000 × g at 4 °C (see
Note 2). - 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. - Place 700 μL of the samples to the isolating column, close the
lid, and centrifuge at 8000 × g for 15 s at room temperature. - Repeat step 8 with remaining samples.
- Add 700 μL wash buffer to the column and centrifuge at
8000 × g for 15 s at room temperature (repeat washing couple
of times). - 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