DV200 Analysis of FFPE RNA Samples with the Fragment Analyzer

Answer

This Information Applies To: 5200, 5300 and 5400 Fragment Analyzer, ProSize software, DNF-471 RNA kit, DNF-472 HS RNA kit 

Issue:

The DV₂₀₀ score is a quality score for evaluating quality of RNA derived from formalin-fixed paraffin-embedded (FFPE) samples established by Illumina Inc. in 2016. It represents the percentage of RNA fragments larger than 200 nucleotides relative to all RNA fragments. The DV₂₀₀ of samples run with Agilent Fragment Analyzer DNF-471 RNA kit or DNF-472 HS RNA kit can be calculated within the ProSize data analysis software.

Steps to follow

This article contains different instructions for 33 cm arrays and 22 or 55 cm arrays.

 Running a Separation with a DV₂₀₀ Method (33 cm array)

1. Download the DV₂₀₀ calculation configuration and method files for the assay you are running: DNF-471 RNA or DNF-472 HS RNA.
2. Unzip the .zip files.
3. Save the configuration files (these have a .ini extension) for the RNA kit or the HS RNA kit in the following location: C:\ProSize data analysis software\Configurations\33cm.
4. Save the method files (.mthds extension) in the following location: C:\Agilent Technologies\Methods\33cm.
5. Select the DNF-471F33 - SS Total RNA 15 nt - FFPE Illumina DV200 or DNF-472F33 - HS Total RNA 15 nt  - FFPE Illumina DV200 method when setting up a new run in the Agilent Fragment Analyzer controller software. 

The DV₂₀₀ is provided under the Smear Analysis tab in the % of total column for each sample in ProSize.

Applying the DV₂₀₀ Metric to a Previously Run Data File (33 cm array)

1. Download and save the configuration files for the RNA kit or HS RNA kit as described in the 'Running a Separation with a DV₂₀₀ Method' section.
2. Open the file that you wish to apply the DV₂₀₀ metric in ProSize. Carry out the following steps (Figure 1):
   
   1. Select the Option tab.
   2. Click the  button next to Edit Configuration.
   3. A second Advance Setting window will open. Click Load.
   4. Open the Configurations file folder 33 cm (navigate to C:\ProSize data analysis software\Configurations\33cm).
   5. Select the desired configuration file.
   6. Click OK>Apply.

The DV₂₀₀ is provided under the Smear Analysis tab in the % of total column for each sample.

Figure 1. Applying the DV200 configuration to an existing data file

 Applying the DV₂₀₀ Metric to a Previously Run Data File (22 or 55 cm array)

Method and configuration files are only available for the 33 cm array. However, If you have a 22 or 55 cm array it is still possible to calculate the DV₂₀₀ by setting up a smear range within ProSize: 

1. Open the data file for which you wish calculate the DV₂₀₀.
2. From the main screen in ProSize, click the  button.
3. Select the Smear Analysis tab and enter 200 as the start size and 20000 as the end size (Figure 2).
4. This smear range can either be applied to all samples within the open file or applied to selected wells.

The DV₂₀₀ is provided under the Smear Analysis tab in the % of total column for each sample.

 Figure 2. Smear Analysis tab in the Set Individual Parameters dialog box

Learn how to effectively maintain and operate your Fragment Analyzer: 5200, 5300, and 5400 Fragment Analyzer System Manual ProSize Data Analysis Software User Manual Agilent DNF-471 RNA kit (15 nt) Quick Guide for Fragment Analyzer Systems Agilent DNF-472 HS RNA (15 nt) Kit Quick Guide for Fragment Analyzer Systems

For Research Use Only. Not for use in diagnostic procedures.

PR7000-8553