Determinants of RNA quality from FFPE samples

Abstract

The large archives of formalin-fixed paraffin-embedded (FFPE) tissue specimens that exist are a highly valuable source of sample material for molecular biological analysis, including gene expression profiling. However, current data on adverse effects of standard pathological practice on the usefulness of biomolecular analytes obtained from such archived specimens is largely anecdotal. Here, we present a systematic examination of the most relevant parameters for integrity and useability of RNA obtained from FFPE samples, including storage time and conditions, fixation time, and specimen size. The results are particularly relevant for any application relying on cDNA synthesis as an initial step of the procedure, such as RT-PCR, and microarray analysis.

Figure 1. Integrity of RNA isolated from fresh and aged FFPE samples.

Rat tissue samples were formalin-fixed and embedded in paraffin. 10 µm sections were cut within 3 days after embedding (T0), or after 1 year storage at the indicated temperatures (RT–room temperature, 20–25°C). Integrity of RNA isolated from individual sections was analysed by capillary electrophoresis. 18S and 28S rRNA bands correspond to 41–43 and 47–50 [s], respectively. The lower marker (marked ‘*’) corresponds to fluorescence intensity of 7–10 [FU].

Figure 2. RT-PCR efficiency with RNA from fresh and aged FFPE samples.

Rat tissue samples were formalin-fixed and embedded in paraffin. 10 µm sections were cut within 3 days after embedding (T0), or after 1 year storage at the indicated temperatures (RT–room temperature, 20–25°C). The isolated RNA was used for one-step RT-PCR with different primer pairs directed against the rat HPRT gene. Amplicon sizes were 128, 208, 404, and 668 nt. All amplicons were amplified with equal efficiency from RNAlater-stabilized rat tissue (pos. Control), and no products were obtained in no template controls (neg. control).

Figure 3. Effect of fixation on RNA integrity and RT-PCR performance.

Rat tissue samples were either stabilized in RNAlater or formalin-fixed, either over night or for 3 days. Fixed samples were embedded in paraffin, and 10 µm sections were cut within 3 days after embedding for preparation of RNA. RNA quality from each sample type was analysed by capillary electrophoresis. 18S and 28S rRNA bands correspond to 42–44 and 49–51 [s], respectively. The lower marker (marked ‘*’) corresponds to fluorescence intensity of 6–8 [FU]. The color bars indicate RNA performance in one-step RT-PCR using different primer pairs directed against the rat RPL4 gene. Amplicon sizes from top to bottom were 1010, 785, 613, 400, 206, and 96 nt. Red indicates no amplification, yellow–weak amplification, green–good amplification.

Figure 4. Effect of sample size during fixation on RNA integrity and RT-PCR performance.

Rat tissue samples were formalin-fixed over night, either as whole organs, or cut to pieces <3 mm thickness. Samples were embedded in paraffin, and 10 µm sections were cut within 3 days after embedding. RNA quality was analysed by capillary electrophoresis. The lower marker (marked ‘*’) corresponds to fluorescence intensity of 8–9 [FU]. RNA performance was tested in one-step RT-PCR using different primer pairs directed against the rat RPL4 gene. Amplicon sizes as seen on the gel images were 96, 206, 400, 613, and 785 nt.

Figure 5. Fragmentation state and RT-PCR performance of RNA from tumor samples.

RNA was isolated from individual 10 µm sections of FFPE tumor samples of different age (1–10 years). Fragmentation state was analysed by capillary electrophoresis (A). RNA performance was tested in 2-step real-time RT-PCR using assays directed against the human TBP gene, using either a mix of random and oligo-dT primers for cDNA synthesis (B), or oligo-dT alone (C).