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RNA Isolation

Isolation from FFPE Tissue (RNA)

Agencourt FormaPure

The Agencourt FormaPure system provides a simple, automation-friendly process for extracting total nucleic acid from Formalin-Fixed, Paraffin-Embedded (FFPE) tissue samples. By utilizing the Solid Phase Reversible Immobilization (SPRI) paramagnetic bead-based technology, the Agencourt FormaPure system does not require vacuum filtration, centrifugation, or organic solvents such as phenol or xylene. Researchers can go from paraffin digestion to total nucleic acid extraction in about four hours (without setup) for 96 samples in a multi-well format utilizing the Beckman Coulter Biomek NXP or FXP Span-8 workstation. Recoveries are consistently higher than competitive techniques.

Overview

  • Consistent, superior yield and purity of RNA or total nucleic acid
  • No centrifugation, vacuum filtration, or solvents required
  • Supports automated or manual processing


Application: Total RNA or total nucleic acid isolation from FFPE tissue samples
Downstream Application Techniques: qRT-PCR, microarray



Process Overview
 



A - B. Digestion of tissue and release of nucleic acids. 1. Binding of nucleic acids to magnetic beads  2. Separation of beads 3.Washing of captured products with 70% ethanol  4.Washing with isopropanol to remove contaminants  5. Washing with 70% ethanol  6. Elution of nucleic acids from the magnetic particles.
 


Recovery of Fragmented Nucleic Acid
 

The fixation of tissue samples in formaldehyde can cause extensive crosslinking of tissue components. Quality of nucleic acid will vary depending on a variety of factors, including how the formalin-fixation process was carried out, age of sample, storage conditions, etc. As seen in Figure 1, nucleic acid from a variety of FFPE sample types and ages was isolated using the Agencourt FormaPure process and successfully amplified by PCR.


Nucleic Acid Recovery
 



Figure 1. Total nucleic acid was isolated from 5 micron sections for each tissue. Elution volume was 40 µL. Two (2) µL of eluted product was used in a 20 µL PCR reaction (45 cycles) to amplify a 300 bp region of the GAPDH Gene. A 10 µL sample was electrophoresed on a 2% agarose gel. Lane 9 contains the negative control. Lane 10 contains a 1 kb ladder.



Consistent RNA Purity
 

The Agencourt FormaPure system provides consistent RNA yield and purity. Table 1 shows data for RNA recovered from five FFPE rat lung biological replicates which were purified using Agencourt FormaPure. Consistent ratios ranging from 1.79 to 1.83 for 260/280 and 1.06 to 1.21 for 260/230 are observed. Agilent* bioanalyzer traces from the same samples show consistent RNA purity and fragment recovery (Figure 2).
 

Rat Lung
Replicates
OD
260/280
OD
260/230
RNA
Concentration
(ng/µL
)
Volume 
(µL)
Amount 
(Ug)
1 1,79 1.21 288.99 35.00 10.11
2 1.83 1.14 439.80 35.00 15.39
3 1.79 1.07 538.57 35.00 18.85
4 1.80 1.10 629.80 35.00 22.04
5 1.80 1.06 513.13 35.00 17.96

Table 1. Beta site data for RNA purified from FFPE rat lung tissues using Agencourt FormaPure.
One 10-micron section was used for each replicate.

 

RNA Purity
 



Figure 2. Agilent bioanalyzer traces of RNA prepared from several FFPE rat lung tissues using the Agencourt FormaPure system. All samples were treated with DNAse I.


Summary
 

The Agencourt FormaPure system can extract both total nucleic acid and total RNA and offers superior yield and purity in an automation-friendly process. This technology can purify nucleic acids from a wide variety of tissue types for utilization in techniques such as SNP genotyping, gene expression, and sequencing. With the utilization of paramagnetic beads, no centrifugation, vacuum filtration or chemical solvents are required. The Agencourt FormaPure system, along with the Beckman Coulter Biomek NXP or FXP Span-8 workstation, helps to answer today's most comprehensive biological questions.

Webinars & Symposiums

  • Biomek i-Series Platforms: Innovation to Improve Genomic Sample Prep

    Zach Smith, MS Senior Applications Scientist, Beckman Coulter Life Sciences

    Click to play  

  • Challenging Samples in Cancer Research: Formalin-Fixed Paraffin-Embedded Tissues

    Kathy Munkvold, PhD Staff Development Scientist Beckman Coulter Life Sciences

    Click to play  

    Increasingly, Formalin-Fixed, Paraffin-Embedded (FFPE) samples are an essential part of many next-generation sequencing (NGS) sample prep workflows, specifically in the field of cancer research. High quality extraction protocols focused on...

  • High-Performance RNA & miRNA Extractions using SPRI

    Bee Na Lee, PhD, Applications Scientist Beckman Coulter, Inc.

    Click to play  

  • Improve Yield and Quality from RNA and miRNA Extractions from FFPE Tissue

    Alexandra Scavo PhD, Sr. Applications Scientist, Beckman Coulter, Inc. and Bee Na Lee PhD, Sr. Applications Scientist, Beckman Coulter, Inc.

    Click to play  

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