Affymetrix miRNA Analysis by GeneChip miRNA 3.0 Arrays.

Judith Sunzenauer (last update 2.4.2013)

Procedures

Poly (A) Tailing:

• Use 1 µg of total RNA and adjust to 8 µL with nuclease-free water.
• Add 2 µL of RNA Spike Control Oligos to each reaction tube.
• Dilute the ATP mix (vial 3) 1:500 with 1mM Tris.
• Prepare the Poly A Tailing Master Mix:

• Add 5µl Poly A Tailing Master Mix to 10µl RNA/Spike Control Oligos. Mix gently (do not vortex) and zip spin.
• Incubate at 37 °C for 15min.
• Spin down and place on ice.
• Add 4 µL 5X FlashTag Biotin HSR Ligation Mix (Vial 5) to each sample.
• Add 2 µL of T4 DNA Ligase (Vial 6) to each sample. Mix gently (do not vortex) and zip spin.
• Incubate at RT for 30min.
• Stop the reaction by adding 2.5 µL HSR Stop Solution (Vial 7). Mix and zip spin.
• Remove 2 µL of the biotin-labeled sample for use with the ELOSA QC Assay. It is acceptable to store the 2 µL of biotin-labeled sample on ice for up to 6 hours or at -20 °C for up to 2 weeks, and run the ELOSA QC Assay at a convenient time.
• The remaining 21.5 µL biotin-labeled sample may be stored on ice for up to 6 hours, or at -20 °C for up to 2 weeks, prior to hybridization on Affymetrix GeneChip miRNA Arrays.

Hybridization:

Set the temperature of the oven to 48 °C and set the RPM to 60 and allow the oven to preheat for at least 1h. Unwrap the Arrays and allow the Arrays to warm to RT. Insert a 20 µL or 200 µL pipet tip (unfiltered type recommended) into the upper right septum for proper venting when hybridization cocktail is injected. Bring the reagents for the Hybridization Master Mix to RT.

• Completely thaw and then heat the 20X Eukaryotic Hybridization Controls for 5 minutes at 65 °C.
• Prepare the Hybridization Master Mix:

• Add 110µl of Hybridization Master Mix to each sample.
• Incubate at 99 °C for 5min, then 45 °C for 5min.
• Aspirate 130 µL and inject into an array.
• Remove the pipet tip from the upper right septum of the array.
• Cover both septa with 1/2″ Tough-Spots® to minimize evaporation and/or prevent leaks.
• Place the arrays into hybridization oven trays.
• Load the trays into the hybridization oven.
• Incubate the arrays at 48 °C and 60 rpm for 16 to 18 hours.

Washing and Staining:

• Remove the arrays from the oven. Remove the Tough-Spots from the arrays.
• Extract the hybridization cocktail from each array and transfer it to a new tube. Store on ice during the procedure, or at -80 °C for long-term storage.
• Fill each array completely with Array Holding Buffer.
• Allow the arrays to equilibrate to room temperature before washing and staining.

Note: Arrays can be stored in the Array Holding Buffer at 4 °C for up to 3 hours before proceeding with washing and staining. Equilibrate arrays to room temperature before washing and staining.

• Place vials into sample holders on the fluidics station:

A. Place one (amber) vial containing 600 µL Stain Cocktail 1 in sample holder 1.

B. Place one (clear) vial containing 600 µL Stain Cocktail 2 in sample holder 2.

C. Place one (clear) vial containing 800 µL Array Holding Buffer in sample holder 3.

• Wash and stain with Fluidics Station 450 using the appropriate fluidics script.
• Check for air bubbles. If there are air bubbles, manually fill the array with Array Holding Buffer. If there are no air bubbles, cover both septa with 3/8″ Tough-Spots. Inspect the array glass surface for dust and/or other particulates and, if necessary, carefully wipe the surface with a clean lab wipe before scanning.

Scanning:

Make sure the laser is warmed up prior to scanning for 15min. Bring the Array to RT.

• On the back of the probe array cartridge, clean excess fluid from around septa. Carefully apply one tough-spot to each of the two septa. Ensure that the spots lie flat.
• Select your experiment and click START button.
• Open the sample door on the scanner and insert the probe array into the holder. The door closes automatically through the User Interface when start scan is selected à click OK.

Materials: