Diagnostic methodologies need per definition a basic level of standardization in order to obtain concordance in the responses among hospitals. Immunohistochemistry (IHC) is the most widely used method to detect proteins in FFPE tissues for diagnostic purposes. Despite the standardization of immunohistochemical staining protocols, some factors could still vary among samples and laboratories, such as tissue fixation (duration), choice of antibodies, and the threshold for interpretation of positive immunostaining, which may dramatically affect test accuracy and reproducibility. In any case IHC is considered an accurate diagnostic method because there are guidelines and inter-laboratory quality controls through the cross-testing of cases that guarantee the reliability of the assays. Nowadays, there is great interest and need for molecular pathology tests in order to better characterize pathologies and better define the individual patient profile for tailored therapy. In the present study, we have systematically compared the results of different homemade and commercial protocols for DNA and RNA extraction on the same set of FFPE tissue specimens used in several laboratories in order to define reliable criteria on nucleic acid extraction from FFPE tissues.
The design of this multicentre study allows determining the specific effects of the different extraction protocols, since the quality control experiments and interpretation of the multicentric results were performed in a single laboratory. Nonetheless, we cannot entirely exclude the possible effects of additional parameters, such as different technicians and laboratory equipments, which may influence the results. However, as the extractions were performed by experienced technicians in molecular diagnostic laboratories, we anticipate a minimal effect of these parameters.
The FFPE tissues used in this study originate from routine diagnostics of three different pathology departments. Hence it may well be possible that fixation time and the size of the tissue pieces used, which is important for the fixation rate, varies. Formalin not only cross-links proteins and nucleic acids, it also modifies RNA by adding mono-methylol groups to all the four bases blocking reverse transcription . However it has been shown that the efficiency of extracting amplifiable RNA and DNA from FFPE tissue is inversely proportional to the fixation time [6, 7] and thus standardized fixation conditions are important. This was not specifically addressed in the current study since all laboratories used serial sections of the same tissue block of each cancer tissue.
DNA extraction protocols that used purification of the extracted DNA, either by precipitation (protocol type 1) or by using silica-based columns (protocol type 3) gave comparable results in terms of yield and purity of the DNA. The apparent yield of DNA by the use of these protocols is less comparable to protocols that do not employ any purification (protocol type 2), which is not surprising, since the measured DNA-concentration in the un-purified samples reflects the presence of RNA and DNA. Yield and concentration from protocols that do not employ purification steps is thus not accurate since degraded RNAs as well as protein debris are also quantified, overestimating thereby the real DNA amount.
The PCR-amplifiability of the DNA samples was not drastically affected by the use of different DNA-extraction protocols. Our study showed that the measurement of amplify-ability of the DNA by performance of the BIOMED-2 control gene PCR is the preferred parameter, superior to spectrophotometrical determination of the A260/A280 ratio, to measure DNA-quality for diagnostic molecular testing. In general, DNA obtained from FFPE samples that allows amplification of 300 bp is considered as good DNA-quality and can be used for many molecular tests, including clonality testing using the BIOMED-2 primers and protocols . Array comparative genomic hybridization (aCGH) was successful using DNA samples from FFPE tissues, which allowed amplifying 200 bp in the BIOMED-2 control gene PCR . Based on evaluation of the DNA-amplifyability, there is not a single DNA-extraction protocol that is highly superior to others. The majority of the DNA-extraction protocols, except for the DNA-extraction protocol by participant 1, give similar and comparable results. There was only a minor difference in outcome, showing less performance of the DNA-extraction protocol used by participants 3 and 8a. However, as this was seen only in one of the four tested samples, there is no solid reason to avoid the use of these protocols.
Upon selection of the protocol to use in a diagnostic laboratory, also the requirements for the specific molecular test should be taken into consideration. It should be realised that molecular tests that require a standardized input of DNA, should use purified DNA-samples, allowing the most accurate DNA-quantification. For complex multiplexed PCRs or tests that require a specific accurate input of DNA per PCR test, like B- /T-cell clonality assessment, , as well as for application of array-CGH , MLPA-based tests , we strongly recommend using type 3 protocols that use silica based adsorption columns for purifying DNA.
For other molecular tests like PCR followed by direct sequencing, however, purification may not be essential. The use of protocols without further purification (protocol type 2) has an additional advantage, it is more rapid and less expensive compared to DNA-protocols that employ purification steps. We therefore recommend the use of a simple DNA-protocol, such as the type 2 protocols (DNA extractions without further purification or precipitation), for general molecular tests as mutation and translocation detection. Whichever the method used, it is highly recommended to employ only optimized and controlled reagents. This can be an important source of result variability.
The RNA isolation methods affected the yield of the extracted RNA, even when extraction conditions (proteinase K incubation time and temperature) were comparable (Supplemental Table 3). Proteinase K digestion is an important step affecting the RNA yield. In fact, we observed that an extended digestion time resulted in a more efficient extraction, especially from colon and ovarian tumour tissue using the Qiagen RNeasy FFPE kit. This is consistent with previous reports demonstrating that increasing proteinase K digestion time improved the RNA yield [6, 10, 11]. Formalin fixation preserves cellular structures by crosslinking biomolecules and thus traps nucleic acids and prevents the extraction of amplifiable RNA and DNA. By extending the digestion time to at least 16 hours, proteinase K is thought to release protein cross-linked nucleic acids more efficiently to permit subsequent RNA/DNA isolation, e.g. by column based methods . Surprisingly, extended proteinase K incubation had no effect on the RNA quality evaluated by RT-PCR.
The RNA quality assessment by spectral photometry did not correlate with the maximum amplifiable length (Tables 1 and 3). The ratio A260/280 is an indicator of possible contaminants  and the presence of inhibitors for subsequent reactions, however, this measurement is highly variable  and does not assess the length of the RNA extracted. Similarly, the widely used quality assessment by capillary electrophoresis (Bioanalyzer or Experion) is not predictive of the utility of RNA samples isolated from FFPE tissue in molecular analyses, since the most limiting factor for RT-PCR and presumably other enzymatic procedures, is the chemical modification of RNA by formaldehyde, which is not resolved by electrophoretic assays [10, 14].
Regarding the high degree of degradation of RNA in FFPE tissues, the ability of amplifying 250 bp PCR-fragments indicates RNA of very good quality. Nevertheless, the successful amplification of target mRNA fragments ≥200 bp in length was considered good RNA quality [11, 15]. All isolation methods resulted in RNA of useful quality for expression analyses in archival and diagnostic tissues, since shorter amplicons are sufficient for quantitative PCR [15–17]. This study reveals that column based methods (protocol type 6) provided best RNA quality assessed by RT-PCR from most of the tissues. Moreover these methods, in comparison to homemade protocols and commercial monophasic solutions, are less time-consuming by omitting the precipitation step to isolate the RNA. Small adjustments of the manufacturer’s instruction (e.g. proteinase K digestion) may have caused inter-laboratory variability both in RNA quantity and quality, which is consistent with earlier reports demonstrating that different results were obtained by different groups using the same commercial kits [10, 15].
Although homemade RNA extraction methods (protocol type 4) or commercial solutions (protocol type 5) may be suitable for certain applications in research and diagnostics using short amplicons (below 160 bp), our results demonstrate that among the analyzed methods , particularly the Qiagen RNeasy FFPE kit including an extended proteinase-K digestion period is recommended. Due to the observed inter-laboratory variability, further standardization of the techniques, including commercial kits, is recommended for research and diagnostic applications in molecular pathology.