Affymetrix miRNA Analysis by GeneChip miRNA 3.0 Arrays.
Judith Sunzenauer (last update 2.4.2013)
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:
|10x Reaction Buffer (Vial 1)||1,5 µl|
|25 mM MnCl2 (Vial 2)||1,5 µl|
|Diluted ATP Mix||1 µl|
|PAP Enzyme (Vial 4)||1 µl|
- 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.
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:
|2x Hybridization Mix||66µl|
|27,5% Formamide (Vial 12)||19,2µl|
|20X Hybridization Controls||6,6µl|
|Control Oligo B2, 3nM||2,2µl|
- 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-SpotsÂ® 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.
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 Ã click OK.
|GeneChip miRNA 3.0 Arrays||Affymetrix||#902017|
|FlashTag Biotin HSR RNA Labeling Kit||Affymetrix||#901910|
|GeneChip Hybridization, Wash and Stain Kit||Affymetrix||#900720|
|GeneChip Hybridization Control Kit||Affymetrix||#900454|
|Hybridization Oven 640||Affymetrix||#800139|
|Fluidics Station 450||Affymetrix||#00-0079|
|GeneChip Scanner 3000G||Affymetrix|