Multimedia User Manual

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Multimedia User Manual for the GEArray Q Series
The Following is a step-by-step manual for the
use of the GEArray Q Series products. Please
refer to the printed user manual for more
detailed information. If you have any further
questions please contact Technical Support at
1-888-503-3187
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The Kit

• Box 1
• GEAprimer Mix, Buffer A
• GEA labeling Buffer, 5X, Buffer BN
• RNase-free H2O
• 10X Stop Solution, Buffer C
• 10X Denaturing Solution, Buffer D
• 2X Neutralization Solution, Buffer E
• GEAhyb Hybridization Solution
• GEAblocking Solution Q

• Box 2 (for non-Rad detection)
• AP-streptavidin
• 5x Washing Buffer, Buffer F
• 1x AP-assay Buffer, Buffer G
• CDP-Star substrate

• Quick Protocol and grid card
• GEArrayAnalyzer software
• User Manual

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Protocol Overview
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4 easy steps in using the Q series GEArray

• Probe synthesis
• Hybridization
• Detection
• Image and data analysis

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1. Probe Synthesis
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Probe Synthesis (Preparing the Annealing Mix)
Total RNA is used as the template (1) Prepare the
annealing mix Total RNA 1-5 mg GEAprimer Mix
(Buffer A) 3 ml add H2O to a final volume 10
ml. Heat the mix at 70ºC for 3 min. Cool the
mix down to 42 C And incubate the mix 42ºC for 2
min
TipsReaction can be carried out on a PCR
machine
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Probe Synthesis (Preparing the Labeling Mix)

• Set up labeling mix
• Warm up the labeling mix to 42 C then mix
• Mix labeling mix with RNA

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Probe Synthesis (Labeling Reaction)
(5) Labeling reaction 42ºC 25 min for 32P
detection 90 min for non-Rad. detection. (6) Stop
the reaction with 2 ml Stop Solution (7)
Denature the probe With 2 ml denaturing
Solution at 68ºC for 20 min. (8) Probe
Neutralization with 20 ml Solution at 68ºC for
10 min.  
Reaction can be carried out on a PCR machine
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2. Hybridization
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Hybridization

•  
• Wet the membrane with H2O
• Prepare hybridization solution
• Pre-hybridization at 60 ºC for 1 to 2 hrs.

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Hybridization (cont.)
4. Mix labeled probe with hybridization buffer,
then add the mix to the pre-hybridized membrane,
incubate overnight at 60ºC
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Hybridization (Washing stage)
Votex the tube after adding the wash solution
5. Wash the membrane twice with 2X SSC, 1 SDS
for 10 min at 60ºC. 6. Wash the membrane twice
with 0.1X SSC, 0.5 SDS for 10 min each at 60ºC.
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3. Detection
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Detection (Radioactive
detection) Option 1 X-ray film and
cassette Option 2 Phosphor-imager
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Detection (Non-Rad. detection)
(1) Block the for 40 min with GEAblocking
solution. (2) Incubate with alkaline
phosphatase-conjugated streptavidin for 30 mins.
 
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Detection (Non-Rad. detection)
(3) Wash the membrane 4x with buffer F followed
by one wash with buffer G. (4) Detecting the
GEArrayTM Q series by chemiluminescence Incubate
membrane with CDP-Star? chemiluminescent
substrate Record the image with X-ray film or a
digital imaging system.
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4. Image and Data Analysis
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Data collection and analysis overview
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Image and Data Analysis

• Record the GEArray image and convert the image
  into an image file.
• Analyze the image with an image analysis program
  such as ScanAlyze or ImageQuant.
• Process and save the raw data table

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Image and Data Analysis
Analyze the raw data table(s) with GEArratAnalyzer