RNA Submission Information

Step-By-Step Instructions For Meeting The Criteria For RNA Submission 

Criteria for RNA approval

·         RNA must be extracted by either TriReagent/Trizol or Qiagen RNAeasy columns.  Qiagen RNA columns are recommended.  

·         An aliquot of RNA must be submitted in 10mM TE for Agilent/Nanodrop analysis at a concentration of 1ug/10ul.

·         The area under the two rRNA peaks be at least 33% of the total area of the RNA profile generated by the Agilent Bioanalyzer.

·         260/280 ration must be between 1.7-2.0.

·         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 RNA submission:

RNA submission checklist

Call (513) 558-4764/4769 if you have any questions

STEP 1

Required: Check the proper box on the RNA Submission Form

• Check the proper box on the RNA Submission Form.

STEP 2

Required:  RNA extraction

• RNA must be extracted by either TriReagent/Trizol or Qiagen RNAeasy columns.  Qiagen RNA columns are recommended. 
• Click here for our recommended RNA extraction protocol.

• Check the proper box on the RNA Submission Form.

STEP 3

Required:  Analysis of RNA by an Agilent 2100 Bioanalyzer/Nanodrop Spctrophotometer.

Bring the following items to Kettering #336:

1.      RNA  

·         1 ug of each RNA in 10ul of 10mM TE, frozen.  Please label clearly all tubes.

·         Bring only the RNA samples that are to be analyzed by the Agilent Bioanalyzer.  

2.      A copy of the RNA submission Form for Analysis  

·         We will notify you when the Agilent Bioanalyzer/Nanodrop RNA analysis is completed.

·         RNA is approved for Microarray work if it meets the following criteria

·         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.

·         If the 260/280 ratio is between 1.7-2.0.

·         Based on the Agilent Bioanalyzer data, we will inform you what RNA amount is required to reach a 2ug/ul RNA concentration in a 10ul total volume. 

·         You must submit RNA for microarray work in nuclease free water at a concentration of 2ug/ul.

·         If the 2ug/ul RNA concentration is not attainable, contact us. 

·         The RNA is approved for submission:  We will repeat the experiments at no charge if the data are not satisfactory.

·         Go to STEP 4 of the check list.

·         See STEP 5 to order microarrays online.

• If the RNA fail to meet our criteria the RNA samples will not be approved for microarray analysis

STEP 4

Required:  Proper Preparation of the RNA for microarray analysis

• 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.

·         If the RNA is >2ug/ul for total RNA or >0.1ug/ul for poly(A)+ RNA, dilute to the required 2ug/ul concentration.

·         If the RNA is <2ug/ul for total RNA or <0.1ug/ul for poly(A)+ RNA, dry down or ethanol precipitate 20ug of total RNA or 1ug of poly(A)+ RNA and resuspend in 10ul RNase-free water.

·         We will not accept RNA samples that are not at the required concentrations.

·         On the RNA Information Table (page 3) of the RNA Submission Form, write down in the "Conc. ug/ul" column the concentration of each RNA sample.

·         On the RNA Information Table (page 3) of the RNA Submission Form, place a checkmark in the "Volume 10ul or more" column if the volume of each RNA sample is 10ul or greater.

·         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.

·         In the "Labeling" column you may specify how each RNA is to be labeled. Leave blank if no preference.

STEP 5

Required:  Place order for microarray analysis online

• Click Here to Order