Agilent

Hybridization protocol for Agilent miRNA Custom arrays

This protocol was developed in collaboration with Dr. Edwin Cuppen, Hubrecht Laboratory, Utrecht, The Netherlands.

This protocol is Agilent's 60-mer in situ synthesized oligonucleotide microarray protocol, Whole Rat Genome 4x44K, adapted for use of the 8x44K format and hybridization of small RNA.
The protocol was tested with small RNA enriched from 1.5 - 3 µg of total RNA per dye.

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A. Preparation of samples

  • Set up hybridization reactions as follows: 

Test sample (Cy3-ULS labeled)

...µl

Reference sample (Cy5-ULS labeled

...µl

RNAse free water

...µl

Total

40 µl
  • Mix well using a vortex
  • Incubate samples at 60°C for 2-3 min
  • Add 40 µl 2x GEx Hybridization buffer HI-RPM (very viscous)
  • Mix well by careful pipetting. Take care to avoid introducing bubbles. DO NOT vortex
  • Spin briefly at 13,000 x g, put samples on ice and use immediately

B. Prepare the gasket slides for slide format 8x44K

Note: Never touch the surfaces of the slides. Never allow the microarray surface to dry out during the hybridization process and washing steps. Protect samples from light.

C. Hybridizing Cy3-ULS and Cy5-ULS labeled samples to slides

  • Procure as many assembled chambers as needed to complete the microarray
  • Disassemble by turning the THUMBSCREW counterclockwise to loosen the CLAMP ASSEMBLY, slide off the CLAMP and remove the CHAMBER COVER
  • Label the CHAMBER BASE with an ID that can be easily matched to the microarray ID
  • Load a clean GASKET SLIDE into the CHAMBER BASE with the LABEL facing up and aligned in the rectangular section of the CHAMBER BASE
  • Ensure that the GASKET SLIDE is flush with the CHAMBER BASE and is not ajar
  • Slowly draw up all the entire amount of solution from a tube, avoiding any bubbles in the solution. Use 60 µl for 8x44K: due to viscosity of the sample, not the entire 80 µL can be used. Make sure that at least 60 µL is used.
  • Touch the pipette tip to the CENTER of the GASKET WELL. Slowly dispense the solution into the CENTER of the microarray GASKET WELL area - avoiding any introduction of bubbles

DO NOT move the CHAMBER BASE or GASKET SLIDE once the hybridization solution has been dispensed.

  • Remove the appropriate Agilent oligo microarray slide from its packaging. Handle the microarray slide only by the ends of the slide to avoid damage
  • Flip the slide so that the NUMERIC SIDE is FACING UP as you lower it onto the GASKET SLIDE. Lower carefully and align with the 4 guideposts on the CHAMBER BASE. Once aligned and slightly above the GASKET SLIDE, gently place the oligo microarray slide against the GASKET SLIDE to complete the sandwiched slide pair. Quickly assess that the slides are completely aligned and that the oligo microarray is not ajar (ends/sides can get caught on the upper part of the CHAMBER BASE)
  • DO NOT move the CHAMBER BASE or sandwiched slides as this can cause leakage of the hybridization mixture
  • Correctly place the CHAMBER COVER onto the sandwiched slides and than slide on the CLAMP ASSEMBLY until it comes to a stopping point in the middle of the CHAMBER BASE and COVER pair
  • Tighten the THUMBSCREW by turning it clockwise until it is fully hand tight.
  • Hold the CHAMBER ASSEMBLY in your hand, VERTICALLY, and slowly rotate it clockwise 3 times to allow the hybridization solution to wet the gasket
  • Inspect the sandwiched slides and note the bubble formation. A large mixing bubble should have formed which can move freely. If bubbles do not move, gently tap one corner of the assembled chamber on a hard surface and rotate it vertically again.
  • Continue loading and assembling the rest of the Agilent microarray hybridization chambers. Up to 4-8 GASKET SLIDES may have hybridization sample dispensed on them at a time prior to adding the microarray slide
  • Once all the chambers are fully assembled, load the hybridization rotator rack and make sure it is balanced
  • Hybridize at 37°C for 17 hrs at 10 rpm (only when using 2x GEx hybridization buffer)

D. Washing the slides

  • Prepare the four following staining dishes: 

i. Add Wash solution 1 (at room temperature) to staining dish 1 (~250 ml volume)

ii. Add a slide rack and a magnetic stir bar to staining dish 2, and add enough Wash Solution 1 (at room temperature) to cover the rack. Place the dish on a magnetic stir plate

iii. Place the third washing dish on a magnetic stir plate. Add a magnetic stir bar to the washing dish. Add Washing solution 2 to a depth sufficient to cover the slide rack

iv. Place the fourth washing dish on a magnetic stir plate. Add a magnetic stir bar to the washing dish. Add Washing solution 3 to a depth sufficient to cover the slide rack

  • Remove only 1 hybridization chamber from the oven at a time to avoid chamber cool-down before disassembly
  • Place the hybridization CHAMBER ASSEMBLY on flat surface and loosen the THUMBSCREW, turning COUNTER-CLOCKWISE
  • Slide off the CLAMP ASSEMBLY and remove the CHAMBER COVER
  • With gloved fingers, remove the 'sandwiched slides' from the CHAMBER BASE by grabbing the slides from their ends Keep the oligo microarray slide (NUMERIC BARCODE FACING UP) as you quickly transfer the sandwiched slides to the first staining dish
  • Without letting go of the 'sandwiched slides', submerge the slides into the first staining dish containing Wash solution 1
    With the 'sandwiched slides' completely submerged in the wash solution 1, pry the two slides apart from the barcode end only

v. Slip the blunt end of a pair of tweezers between the slides and GENTLY turn the tweezers upwards or downwards

vi. Let the GASKET SLIDE drop to the bottom of the staining dish

  • Remove the oligo microarray slide quickly and place it into the slide rack contained in the second staining dish, also containing wash solution 1 at room temperature. Minimize exposure to air. Touch only the bar-coded portion of the slide or its edges
  • Repeat steps for all other chambers
  • After all slides have been collected in the slide rack in washing dish 2, set the magnetic stir plate to maximum speed (setting 4) and wash for 1 minute
  • Transfer the slide rack to the third staining dish, which is, containing wash solution 2. Place the entire dish on a magnetic stirring plate set to medium speed (setting 4). Wash the slides for 1 minute. Not longer, not shorter
  • Place the rack on some blotting paper to get rid off some unnecessary fluids
  • The slide rack needs to stay immersed in the wash solution 3 during the individual slide drying process for 30 seconds Remove the rack slowly from the fluid (take 10 seconds for this) and let the slides dry (wash all the dishes while letting the slides dry)
  • The dried slides are ready to be scanned in a microarray scanner

vii. Place slides in 'holders': active side up

viii. Load carousel

ix. Run program (Agilent scan control)

x. Check the settings for 8x44K

  • After scanning, store the slides in polypropylene slide boxes without cork or foam inserts, in a vacuum desiccator or a nitrogen purge box, in the dark. Storage under nitrogen is recommended

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