This protocol is for a microRNA profile using the miRCURY™ LNA Arrays with the Tecan HS4800™ Pro Hybridization Station. Protocols for manual hybridization are available at Web site (external).
Before starting the experiment:
Use the miRCURY™ LNA microRNA Power labeling kit for labeling of your sample(s).
Dissolve the Spike-in miRNA in 30 µl of nuclease free water. Leave the suspension on ice for 30 minutes to dissolve. Vortex and then spin to collect tube contents. Store the dissolved spike-in miRNA at −20° C until use.
Please refer to the instruction manual of your hybridization station for correct volume of buffers required to perform the hybridization. The volumes in Table 1 applies to the hybridization of 4 slides in a Tecan HS400/HS4800 hybridization station. Protocols for various automated hybridization stations are available at Web site (external). Protocol:
- Combine the labeled sample(s) The two samples from the Hy3TM and Hy5TM labeling reactions are combined on ice. Total volume should be 25 µL.
- Add 25 µL Hybridization buffer Check for precipitation (see p. 12) in the Hybridization buffer before adding 25 µL to the labeled sample(s). Mix by vortexing and spin briefly.
- Incubate at 95° C for 2 min. During the incubation the target preparation should be protected from light.
- Incubate 2 min. on ice Leave on ice for at least 2 min. and up to 15 min. Briefly spin the reaction after ice incubation.
- Flush hyb. chamber with 1x Hybridization buffer Check the appropriate volume of the chamber in the suppliers manual and add 1x diluted Hybridization buffer. Dilute with water.
- Inject reaction mixture Inject the 50 µL target preparation to the hybridization station.
- Incubate at 56° C for 16 h. Set the program for the hybridization station to 56° C and 16 h. incubation. Agitation should be set to medium, if possible.
- Two runs of wash at 56° C for 1 min. using Wash buffer A Set the program for the hybridization station accordingly.
- Two runs of wash at 23° C for 1 min. using Wash buffer B Set the program for the hybridization station accordingly.
- Two runs of wash at 23° C for 1 min. using Wash buffer C Set the program for the hybridization station accordingly.
- Wash at 23° C for 30 sec. using Wash buffer C Set the program for the hybridization station accordingly.
- Dry slides Set the program for the hybridization station accordingly.
Total handling time: 1 hour
