SHAPE

RNA MoleculeThe Facility is pleased to announce a new service - Selective 2’-Hydroxyl Acylation analyzed by Primer Extension (SHAPE). SHAPE is a method of RNA analysis that is used to determine RNA structure at the nucleotide level. RNA molecules are combined with a hydrolyzing agent, such as benzoyl cyanide (BzCN), which selectively hydrolyzes unpaired nucleotides, forming 2’-O-adducts. RNA molecules then undergo a primer extension reaction, which the adducts inhibit, forming cDNA fragments that terminate at the location of hydrolysis. These fragments are analyzed on the 3730 DNA Analyzer (Applied Biosystems, Inc.) along with a negative control and one or two dideoxy sequencing reactions. The electropherograms from these reactions are aligned with each other, as well as a reference sequence, to determine the identity of unpaired nucleotides in the original RNA molecule.

Please consult the PDF icon Procedures and Recommendations for SHAPE analysis [pdf] for complete instructions.

Analysis Software

The Facility has the following software to aid in the analysis of SHAPE data generated by the 3730 DNA Analyzer (*.fsa files): GeneMapper and QuSHAPE.  In addition, there are several free programs available for analyzing the electropherogram files with varying levels of features:

  1. Xplorer (Windows or Mac; also part of dnaLIMS)
  2. PeakScanner (Windows)
  3. STRand (Windows)
  4. PeakStudio (Mac or Windows)
  5. RNAstructure (Mac, Windows, or Linux)
  6. And many more.

Order

How to place a order:  

  1. Go to dnaLIMS and login
    (or create an account if you have not done so previously)
  2. Create a Panel for your order(s). This can be edited in the future, and will be available for all future orders. Panels are unique to each individual customer.
    1. Select the “Genetic Marker Editor” on the left of the page, under the heading “Fragment Analysis”.
    2. Check the circle next to “Create Entry” and select “Submit”.
    3. Enter a name for your panel (such as SHAPE), select MS from the dropdown menu, and enter the number of markers (fluorophores). You should have at least three markers (Positive, negative, and ddNTP); the Marker editor will accept up to 8. Click “Submit”.
    4. Enter your Marker Names and the corresponding Dyes and size ranges. Select unknown for Repeat #, and select LIZ600 for Size Standard.
    5. Click on “Finalize”.
  3. Select “Menu” from the top of the page to return to home.
  4. Select “96-and-48 Well Plate Requests” under the heading “Fragment Analysis
  5. Follow the instructions at the top of the page to fill out a spreadsheet containing your sample names. Sample names here must match the labels on any tubes submitted. If submitting in a 48- or 96-well plate, make sure that the plate wells correspond with the well designations on the spreadsheet.
  6. Select “MS” from the dropdown menu entitled “Select Service Type”.
  7. Select your option for “Data Analysis Required”.
  8. Verify all billing information is correct.
    1. OSU customers, you can use this order form to generate a cost estimate, submit your eRequest, and then return to dnaLIMS to update the PR number if required.
  9. Select “Browse…” and navigate to the file created in step “e”. Select “Open” or double click on the file.
  10. Select “Submit”.
  11. Contact PMGF if you have any issues.

How to deliver samples

How to get results:

  1. An email will be sent to you when the .fsa (electropherogram) files are available.
  2. Log into dnaLIMS and click on "Download Fragment Analysis Results".
  3. For electropherogram analysis results an email will be sent to you and then log into dnaLIMS and go to the "Download" tab.

[pdf] - Some links on this page are to .pdf files. These are designated by [pdf] following the link. If you require these materials in alternate format contact our web manager.

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