RNA Isolation from FFPE Tissue 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  Recovery of Fragmented Nucleic Acid

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.

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).

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.