265- Mix and centrifuge.
- Dispense 1.0 μL (or alternative according to the kit manual) of
20× MicroRNA Assay mix into the corresponding PCR reac-
tion tube. - Transfer above into corresponding wells.
- 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. - Mix and centrifuge.
- 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). - Transfer the results to Excel or an alternative spreadsheet and
analysis software.
4 Notes
- Measure the tissue weight. Do not take more than 50 mg of
tissues and do not thaw tissues before adding lysis reagents. - 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. - 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. - 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. - You can leave on ice for a minimum of 2 min and a maximum
of 15 min. - Make sure the sample is protected from light to not interfere
with the dye. - 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. - 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. - 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