263- Denature by incubating at 95 °C for 2 min and immediately
cooling down on ice (see Note 8). - Put the backing slide in the chamber base.
- Add 400 μL of the sample mixture onto the gasket slide (see
Note 9). - Place a microarray slide on top of the backing gasket slides
with the array side facing the target samples (see Note 10). - Clamp the array/backing slide into the hybridization
chambers. - Incubate at 56 °C for 16 h in a hybridization oven with
rotation. - 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. - 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. - Wash the slide for 2 min in the first wash buffer at 56 °C.
- Rinse the slides with the second wash buffer at room tempera-
ture containing 20 mL 20× salt buffer and 380 mL nuclease-
free water. - Wash the slide for 2 min in the second wash buffer and at room
temperature in a new jar. - 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. - Remove the slides gently allowing the buffer to run off.
- Transfer the slide rack to a new staining dish with 99% ethanol
at room temperature for a few seconds. - Remove the slides gently allowing the wash to run off.
- Quick-dry the slides by centrifuging at 600 × g for 2–5 min.
- Scan slides immediately after drying.
- 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 include3.3 Quantitative
Real-Time Polymerase
Chain Reaction
(RT-qPCR)
MicroRNA Detection and Esophageal Adenocarcinoma