School of Medicine

Wayne State University School of Medicine

RNA Isolation

 

General Information

Total RNA isolation services are available for cultured cells, blood, fresh frozen tissue, and formalin-fixed paraffin embedded (FFPE) tissue. Cells and tissue are disrupted and homogenized in QIAzol™ Lysis Reagent (Qiagen), nucleic acids retrieved by Chloroform extraction, and total RNA purified using the Qiagen EZ1® Advanced. Tissues that require more vigorous physical manipulation are homogenized using the Qiagen TissueLyser II®.

Total RNA isolation from blood is performed using the PAXgene™ Blood RNA System (PreAnalytiX). This method requires specific sample collection tubes available from PreAnalytiX and specific handling instructions.

FFPE samples are processed using the RecoverAll™ Total Nucleic Acid Isolation Kit (Ambion, AM1975)

NOTE:

For miRNA (micro RNAs), the AGTC currently employs a high-throughput, automated method which requires a minimum of 96 samples. Please inquire for specific details regarding miRNA sample requirements and options.

Sample Requirements -Total RNA

All samples submitted for RNA isolation should be prepared as described in the following flowchart . Cells and tissues must be submitted in an appropriately labeled, Eppendorf BioPur Safe-Lock 2.0 ml microcentrifuge tube (cat.# 022600044). If you do not have access to these tubes, the AGTC will supply them for you. Blood must be submitted in PAX Blood RNA  collection tubes.

FFPE sections (8 x 10µm curls) must be submitted in flip-top, nuclease-free, 1.5 ml microcentrifuge tubes. Samples can be submitted as collected, or 1 ml of Histoclear II can be added to each tube. Please note: The AGTC does not use Xylene. Samples containing Xylene will be rejected and returned.

All Cell/Tissue/Blood samples must be stored at -80°C and transported to the AGTC on dry ice. FFPE at room temperature.

Requesting RNA Isolation in iLabs

All AGTC services must be requested in iLabs https://waynestate.corefacilities.org/service_center/show_external/2923

If you need assistance registering with iLabs, please contact Ginette Borovicka.

Log in to iLabs and navigate to the AGTC core page. Select the AGTC Service Request tab. Please ensure that you are requesting services under the correct financial account information for your submission. Select RNA Isolation, then select either RNA Isolation Estimate for a cost estimate for future projects/grants, or RNA Isolation Request for projects ready for submission. Use the Project Description window to enter the projects details (ie. type of sample, number of samples, etc.) Using the Upload button below the window, upload a full sample list prepared in MS Excel. As a simple guideline, it is acceptable to label all sample tubes in numerical order, 1 to n , and indicate the corresponding proper sample ID in the sample list. When this process is complete, click save completed form.

Internal (WSU) Users: An estimated cost for requested services will be entered and submitted for your approval prior to sample submission. Once projected costs are approved by the researcher, the AGTC will initiate sample processing.

External Users: Following approval of the projected cost, a copy of a valid Purchase Order must be uploaded to your project page in iLabs before the AGTC will proceed with sample processing.

Important Notes

  1. RNA isolation services will include RNA quality assessment (except FFPE) unless otherwise indicated by the client.
  2. If you are submitting samples that require RNA isolation in conjunction with Microarray services, please proceed directly to the Microaray Request section and just include RNA isolation when entering the project details. For all other RNA services, RNA isolation will need to be requested separately.

 

Additional Information

If you require assistance entering your service/estimate request in iLabs, further information regarding RNA isolation services, or project consultation, please contact Dan Lott .