| Step-By-Step
Instructions For Meeting The Criteria For Guaranteed Work |
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Guaranteed
Work: If your RNA is approved
and the data are not satisfactory, we
will repeat the microarrays free of charge
Criteria
for RNA approval
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RNA must be extracted
by either TriReagent/Trizol or Qiagen RNAeasy columns.
Qiagen RNA columns are recommended.
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An aliquot of RNA must
be submitted in 10mM TE for Agilent/Nanodrop analysis at a
concentration of 1ug/10ul.
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The area under the two rRNA
peaks be at least 33% of the total area of the RNA
profile generated by the Agilent Bioanalyzer.
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260/280 ration must be
between 1.7-2.0.
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RNA submitted for
microarray analysis must be in nuclease free water at a
concentration of 2.0ug/ul. Do not use DEPC water.
Download
and complete the following form for meeting the requirements for
guaranteed work:
Call
(513) 558-4764/4769 if you have any questions |
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STEP
1 |
Required: Check whether microarray
work is Guaranteed or Non-Guaranteed |
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Guaranteed or
- Non-Guaranteed
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- Check the proper box on the RNA Submission Form.
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STEP
2 |
Required:
RNA extraction |
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- Check the proper box on the RNA Submission Form.
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STEP 3 |
Required:
Analysis of RNA by an Agilent 2100 Bioanalyzer/Nanodrop
Spctrophotometer. |
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Bring
the following items to Kettering #336:
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RNA
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1 mg of each RNA in
10ml of 10mM TE, frozen. Please label clearly all
tubes.
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Bring only the RNA samples that
are to be analyzed by the Agilent Bioanalyzer.
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A copy of the RNA submission Form for Analysis
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We will notify you when the Agilent Bioanalyzer/Nanodrop RNA analysis is
completed.
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RNA
is approved for Microarray work if it meets the following
criteria
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If the area under the two
rRNA peaks is at least 33% of the total area from the
total RNA profile, based on Agilent bioanalysis.
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If
the 260/280 ratio is between 1.7-2.0.
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Based on the Agilent Bioanalyzer data,
we will inform you
what RNA amount is required to reach a 2mg/ml
RNA concentration in a 10ml total volume.
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You
must submit RNA for microarray work in nuclease free water at a
concentration of 2ug/ul.
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If
the 2mg/ml
RNA concentration is not attainable, contact us.
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The
RNA is approved for guaranteed microarray work:
We will repeat the experiments at no charge if the data
are not
satisfactory.
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Go to STEP
4 of the check
list.
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See STEP 5 to order microarrays online.
- If the RNA fail to meet our criteria:
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The RNA
samples will not be approved for
microarray analysis.
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We will
carry out the microarray experiments after consultation
with the investigator; however, the microarray work will
not be guaranteed. Click
here to order non-guaranteed microarrays.
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STEP 4 |
Required:
Proper Preparation of the RNA for
microarray analysis |
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- Total RNA is labeled more efficiently with Cy-3 and Cy-5
than is poly(A)+ RNA and is the preferred form.
- For each labeling reaction, template RNA must be frozen in 10ul of RNase-free water at the concentrations listed below.
| RNA Type |
Concentration (mg/ml) |
Volume / Reaction |
| Total |
2.0mg/ml |
10ml (20mg) |
| Poly(A)+ |
0.1mg/ml |
10ml (1mg) |
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If the RNA is >2mg/ml
for total RNA or >0.1mg/ml for poly(A)+ RNA, dilute to the
required 2mg/ml concentration.
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If the RNA is <2mg/ml
for total RNA or <0.1mg/ml for poly(A)+ RNA, dry down
or ethanol precipitate 20ug of
total RNA or 1mg of poly(A)+ RNA and resuspend in
10ml RNase-free water.
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We will
not accept RNA samples that are not at the required
concentrations.
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On the RNA Information
Table (page 3) of the RNA Submission Form,
write down in the
"Conc. mg/ml"
column the concentration of
each RNA sample.
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On the RNA Information
Table (page 3) of the RNA Submission Form,
place a checkmark in the "Volume 10ml or more"
column if the volume of each RNA sample is 10ul or
greater.
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Two RNA samples are used per slide.
On the RNA Information Table (page 3) of the RNA
Submission Form, write down in the "RNA
Samples" column the names of the RNA samples that
are to be used for a given slide.
- Place a selected title for the experiment in the
indicated box. Place a checkmark in the appropriate
box if your
experiment is to be carried out in triplicate.
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STEP 5 |
Required: Place order for microarray analysis
online
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For
Information About Non-Guaranteed Work
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