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protocol
MicroRNA Profiling Using miRCURY™ LNA Array
Sponsored,vendor-submitted protocol    Sponsored by Exiqon A/S    Published in November 2008 (p.65) DOI: 10.2144/000112667

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:

  1. 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.
  2. 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.
  3. Incubate at 95° C for 2 min. During the incubation the target preparation should be protected from light.
  4. 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.
  5. 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.
  6. Inject reaction mixture Inject the 50 µL target preparation to the hybridization station.
  7. 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.
  8. Two runs of wash at 56° C for 1 min. using Wash buffer A Set the program for the hybridization station accordingly.
  9. Two runs of wash at 23° C for 1 min. using Wash buffer B Set the program for the hybridization station accordingly.
  10. Two runs of wash at 23° C for 1 min. using Wash buffer C Set the program for the hybridization station accordingly.
  11. Wash at 23° C for 30 sec. using Wash buffer C Set the program for the hybridization station accordingly.
  12. Dry slides Set the program for the hybridization station accordingly.

Total handling time: 1 hour

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