Resources Cytosure Hero

Array tips & troubleshooting

What's wrong with my arrays?

Identify and resolve common problems that occur during the array protocol.

Key
Problem
Identifier
Solution

Low A260/280 values Show solutionHide solution

Identifier

Protein contamination

Solution

Re-purify samples using proteinase steps

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1. DNA Sample QC Main Image

High A260/280 values Show solutionHide solution

Identifier

RNA contamination

Solution

Ensure that your DNA extraction protocol includes RNase

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1. DNA Sample QC Main Image

Low A260/230 values Show solutionHide solution

Identifier

Contamination of salts or solvents (e.g. Phenol)

Solution

Re-purified by ethanol precipitation
Resuspending the DNA in TE buffer

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1. DNA Sample QC Main Image

Inaccurate sample concentration Show solutionHide solution

Identifier

High DNA concentration (>350ng/μl)

Solution

Dilute DNA 1:2 in water or suitable buffer

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1. DNA Sample QC Main Image

Low dye incorporation – (poor pmol/μl or DNA concentration values) Show solutionHide solution

Identifier

The wrong temperatures or times are being used
Incorrect volumes used in mastermix preparation
Insufficient mixing of samples, reagents and mastermixes
Too much exposure to light or air
Loss of solution from evaporation

Solution

The wrong temperatures or times are being used

Check temperatures with a calibrated thermometer
Check incubation times against protocol

Incorrect volumes used in mastermix preparation

Check correct volumes are being added
Check pipettes are calibrated correctly

Insufficient mixing of samples, reagents and mastermixes

Gently vortex all reagent tubes (except Klenow)
Flick mix Klenow tube
Briefly spin to drive contents off tube walls

Too much exposure to light or air

Use a closed thermal cycler with heated lid

Loss of solution from evaporation

Use PCR machine with a heated lid
If using tubes, make sure lids are tightly closed
If using plates, use caps not a plate sealer

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2. Labelling Reaction Main Image

Black holes on array Show solutionHide solution

Identifier

Low volume of Hybridization solution

Solution

Ensure the correct volume of hybridization solution has been used
Check no leakage of hybridization solution has occurred

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3. Black Holes On Array Main Image

Non-uniform signal intensities Show solutionHide solution

Identifier

Split, deformity or crack in backing slide

Solution

Check the backing slide seal is intact and has not cracked
Report any gasket slide failures to our support team

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4. Non Uniform Signal Intensities Main Image

Bubble scarring/scotching Show solutionHide solution

Identifier

Jig assembly untouched for too long after hybridization
Oven rotation malfunction

Solution

Check oven rotators are working
Remove jigs from oven one at a time
Disassemble under wash buffer rapidly

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5. Bubble Scarring Main Image

Fluorescent smears across the slide Show solutionHide solution

Identifier

Wash-step contamination with fluorescent material
Dried-out arrays during the hybridization or wash steps

Solution

Ensure dishes are regularly cleaned with appropriate solvent
Ensure clean gloves, forceps and dishes
Carry out additional acetonitrile wash for 1 minute at room temperature

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6. Fluorescent Smears Across The Slide Main Image

High background signal Show solutionHide solution

Identifier

Wash-step contamination with fluorescent material
Wash conditions not stringent enough

Solution

Ensure dishes are regularly cleaned with appropriate solvent
Ensure clean gloves and forceps
Check stirrer is producing a vortex prior to adding slides in wash buffer
Check temperature of oven and washes

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7. High Background Signal Main Image

Poor signal intensity Show solutionHide solution

Identifier

Overly stringent wash or hybridization conditions
Cy5-labelled DNA was exposed to light

Solution

Check protocol for correct wash instructions
Cover tubes with foil or use amber tubes
Check temperature of oven and washes

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8. Poor Signal Intensity Main Image

Low Cy5 signal towards the edges of a feature Show solutionHide solution

Identifier

Wet ozone: outer edges of features dry quicker than inside, exposing edges to ozone

Solution

Ensure slides are scanned immediately after washing
Enclose scanners in a box with ozone scrubbers

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9. Low Cy5 Signal Main Image

Low Cy5 signal gradient with more signal loss at one end of the slide Show solutionHide solution

Identifier

Dry ozone: Degradation during scanning, with exposed end degrading quicker

Solution

Ensure slides are scanned immediately after washing
Enclose scanners in a box with ozone scrubbers

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9. Low Cy5 Signal Gradient Main Image

Array tips & troubleshooting

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