Factors in tissue handling and processing that impact RNA obtained from formalin-fixed, paraffin-embedded tissue

Abstract

Formalin-fixed, paraffin-embedded (FFPE) tissue is the most common specimen available for molecular assays on tissue after diagnostic histopathological examination. RNA from FFPE tissue suffers from strand breakage and cross-linking. Despite excellent extraction methods, RNA quality from FFPE material remains variable. To address the RNA quality factors within FFPE tissues, we studied RNA quality, isolating individual elements of the tissue fixation and processing including length of fixation in formalin and the type of buffer incorporated in the fixative. We examined the impact of the length of the tissue processing cycle as well. The optimal fixation period of 12-24 hr in phosphate-buffered formalin resulted in better-quality RNA. Longer tissue processing times were associated with higher quality RNA. We determined that the middle region of gene suffers less damage by these processes as shown by real-time quantitative RT-PCR. These data provide key information for the development of methods of analysis of gene expression in archival FFPE tissues and contribute to the establishment of objective standards for the processing and handling of tissue in surgical pathology. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Figure 1

Diagram of the pathway a tissue specimen takes from removal from patient to an archival tissue block. After devitalization of the specimen, there is a variable length of time before the specimen is placed in fixative. During this period, called “warm ischemia,” the tissue lacks oxygen and anoxic metabolic pathways take over. The specimen undergoes some form of preparation before fixation, depending on the size and complexity of the specimen. The specimen fixes, optimally in 10 volumes of fixative for a duration suitable for complete penetration of the fixative (1 mm/hr). After fixation, the specimen undergoes “tissue processing” on an automated instrument that serially replaces the fluids within a retort to accomplish the replacement of water with paraffin. This three-step process (dehydration, clearing, and impregnation) is accomplished with a variable number of steps (12–14), with more steps devoted to dehydration than clearing or impregnation. Some steps may contain the same reagent, in an effort to complete the exchange of solvents. Most tissue processors operate under vacuum. After tissue processing, the specimen is “embedded” (surrounded with paraffin that acts as a solid support for microtomy). Conditions examined in this study are summarized within the text boxes. For the experiments in this study, the variables were studied in isolation. For experiments on warm ischemia, RNA was assayed before fixation and processing. In all experiments on fixation and tissue processing, warm ischemia was minimized, with a maximum duration of 15 min. For the experiments on fixation, tissue was processed, fixed in phosphate-buffered formalin, and processed at 30 min/step. For experiments on fixative buffer, fixation time was fixed at 24 hr, and tissue was processed for 30 min/step. For experiments on tissue processing, tissue was fixed for 24 hr in phosphate-buffered formalin.

Figure 2

The profiling of total RNA extracted from warm ischemia time conditions by microcapillary electrophoresis. We presented electropherograms based on each condition; 4C (A), room temperature (B), 37C (C), and RNAlater at room temperature (D). RNA profiles represented relative RNA yield (E) and relative rRNA ratio (F), respectively. Fresh rat kidney was used as a control, generating an rRNA ratio of 1.42 ± 0.03. Quantities and qualities of RNA recovered are expressed in reference to fresh rat kidney (1.00).

Figure 3

Assessments of RNA profiles according to fixation times. We analyzed RNA quality using the Agilent 2100 bioanalyzer, using 200 ng of total RNA extracted from mouse kidney formalin-fixed, paraffin-embedded (FFPE) tissues after 0, 12, 24, 36, 48, or 72 hr of fixation. Representative data are presented as an electropherogram (A). To measure quantitatively RNA degradation, we performed QuantiGene assays with CDK4 and GAPDH gene-specific probe sets using the QuantiGene reagent system (Panomics). Relative expression signals of both genes are normalized to that of frozen kidney (B). Data are the mean of three independent experiments.

Figure 4

RNA quality and gene expression profiles from different fixative buffers. Total RNA was extracted from mouse kidney. Tris-, phosphate-, and CaCl₂-buffered formalin and unbuffered formalin-fixed tissues were coupled with paraffin-embedded processing. The total RNA samples (200 ng) were analyzed on an RNA LabChip using the Agilent 2100 bioanalyzer and are shown here as an electropherogram (A). CDK4 and GAPDH gene expressional profiles were measured with 500 and 200 ng of total RNA extracted from those samples, respectively. Relative expressional signal of both genes are normalized to that of frozen kidney (B). Data are the mean of three independent experiments.

Figure 5

RNA quality profiles from different tissue processing times. Representative data are presented as an electropherogram (A). Relative expressional signals of CDK4 and GAPDH genes are normalized to that of frozen kidney (B). Frozen mouse kidney tissue RNA was used as a positive control. Data are the mean of three independent experiments.

Figure 6

Gene-target optimization for amplification of GAPDH and HPRT mRNA from fresh, FF, and FFPE tissue RNA isolates. Real-time quantitative RT-PCR was performed in three different target regions [5′ and middle region of open reading frame (ORF) and 3′ untranslational area of target gene] with gene-specific probe sets (see Supplemental Table 1), designed by Primer Express software (Applied Biosystems). The x-axis shows the region that is amplified for the target gene. The y-axis shows expression values for targeted RNA regions (log scale). Fresh rat kidney tissue RNA was used as a high-quality RNA control, and relative expression levels of each sample are normalized to the fresh tissue RNA. Data are from three independent experiments and are expressed as mean ± SD.

Figure 7

The hypothetical basic model of RNA quality depending on FFPE tissue processing. The darker gray color represents conditions associated with better RNA quality from FFPE tissue.