Biopsies and surgical specimens are routinely fixed in formalin and embedded in paraffin for histological analysis. Although the morphological integrity of the tissues is preserved, this procedure causes degradation of nucleic acids, predominantly RNA. The development of protocols for RNA extraction from these samples makes it possible to molecularly study long-term archived tissues, opening new perspectives for research and diagnostic investigation. Soguero et al. were able to extract RNA in more than 20 year-old archival liver tissue . In breast cancer, long-term follow-up is important because the time to developing distant metastases and death can vary greatly, with both recurrence time and death extending beyond 15 years after surgery [15–17]. Even patients with small tumors (stage T1N0M0) may have 10-year relapse-free survival rates of less than 75% in the absence of systemic therapy .
In addition to the impact of long-term storage, RNA can also be degraded during the relative delay associated with putting the specimen in fixative following surgical excision and by prolonged fixation prior to paraffin embedding . However, the greater challenge is that RNA is chemically modified during formalin-fixation. Fortunately, some of the alterations can be reversed by heating the RNA to 50–55°C . Many attempts have been made to avoid these chemical modifications by proposing use of alternative fixatives such as Bouin, Carnoy, acetone, and alcohol as substitutes for formalin [5, 10, 20]. These fixatives, however, introduce tissue artifacts that can make microscopic histopathological analysis difficult or even impossible. Moreover, these fixatives are not suitable for immunohistochemistry reactions, much more common in pathologic practice than gene expression analyses. Finally, samples in most archives are already fixed in formalin. So, for retrospective studies, it is imperative to develop and/or refine protocols for RNA extraction from FFPE.
The first step in RNA extraction from FFPE samples is deparaffinization of the sections obtained from the paraffin blocks. When deparaffinization is incomplete, the extracted RNA quality is worse . In our work, in the set of 10-year-old samples, both the quantity and quality of RNA extracted with the deparaffinization solution d-limonene (protocols 2 and 3) were higher than that obtained using xylene – also known as xylol, dimethylbenzene, or methyltoluene – (protocol 1) (Tables 3 and 5). Due to other protocol differences, we cannot assess the impact of the deparaffinizing agent alone. It is worthy to note, however, that with the set of recently archived months old samples, the differences between protocols is smaller (Tables 4 and 6).
Historically, RNA integrity has been evaluated using agarose gel electrophoresis stained with ethidium bromide. Typically these gels show two peaks that correspond to ribosomal RNA species 28S and 18S. According to this technique, a given RNA sample is considered of good quality when the relation 28S:18S is equal to or higher than 2 . The 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) is a microfluidic platform that uses electrophoretic separation of RNA by molecular weight and provides laser-induced fluorescence measurements. Using electropherogram curves, 1208 RNA samples from different sources and in different degrees of degradation were analyzed and an algorithm was created to determine RNA integrity number (RIN). The RIN for a given sample ranges from 1 to 10, from totally degraded RNA to completely intact RNA . The RIN method is superior to the 28S/18S ratio method for evaluating RNA quality in breast cancer tissue .
In frozen tissues, a RIN ≥ 6 provides more reproducible microarray results . In FFPE samples, however, the RIN is much lower because RNA continues to degrade over time into small fragments. However, even low RIN RNA may still be used for some molecular analyses. Fragments of only 60 bp have been shown to successfully amplify in 80% of real-time RT-PCR reactions; even with FFPE samples stored long-term . RNA extracted from 2- to 8-year-old FFPE produces RNA of sufficient quality for microarray analysis in at least 24% of unselected FFPE samples .
In frozen breast cancer tissue Strand et al. suggested a minimum RIN of 6 for gene expression analysis . However, according to Madabusi et al., RNA with a RIN as low as 1.4 have been successfully used for gene expression analysis . In our set of ten year old samples, using a minimum RIN of 1.4 as a threshold for RNA quality, only protocol 2 extracted RNA with a minimally acceptable quality for all 14 samples analyzed, 100% (Table 1). Protocol 1 extracted minimally acceptable RNA in 12 of 14 cases, 86% (failing to isolate minimally acceptable RNA for samples 6 and 7, even with three attempts). Protocol 3 extracted minimally acceptable RNA in 13 of 14 cases, 93% (failing for sample 7, even with three attempts). Protocol 4 extracted minimally acceptable RNA in 13 of 14 cases, 93% (failing for sample 4, even with three attempts) (Table 1). In the set of recently archived months old samples (Table 2), however, it was possible to extract RNA with a minimally acceptable quality in all 14 samples analyzed with all four protocols. These data indicate that ten year old RNA, and specifically RNA that is biologically assayable, could be isolated from every sample by at least one and usually most of the protocols. These data indicate that, if a chosen protocol fails to extract RNA from a given case in a first attempt, another attempt should be tried. However, in our series, if two initial attempts were unsuccessful, a third attempt using the same protocol was less likely to yield biologically usable results, especially for older samples. Thus, we recommend switching RNA isolation protocols after a second failed attempt. It would be unusual to completely exclude a case from molecular analysis because of insufficient or poor quality RNA.
Another important observation can be made in a careful analysis of Tables 1 and 2. There are discrepancies in both quantity and quality of extracted RNA among individual cases for each of the protocols. For example, in sample 7, with protocol 1, test 1, it was possible to extract 160,254 pg/μl of RNA. The quality of this RNA, however, showed complete degradation, without a measurable RIN. On the other hand, in sample 3, with protocol 1, test 1, only 1,236 pg/μl was extracted, but the RIN was 2.3. These data corroborate the observation made by Chung et al. that quantity and quality are independent parameters in RNA extraction .
For all parameters analyzed, both for quantity and quality, Protocol 1 did not perform as well as other protocols on our ten year old archived breast tissue, although it still produced sufficient RNA from most samples (Tables 1 and 3). Of the remaining three protocols (Table 3), protocol 2 extracted slightly higher quality RNA (RIN mean/median: 2.5/2.3) than protocols 3 and 4 (RIN mean/median: 2.3/2.2 and 2.4/2.2, respectively). However, protocol 4 extracted more RNA than the other protocols (mean of 107,042 pg/μl and median 86,682 pg/μl) (Tables 1 and 3). Protocol 4 used a total of 50 μm of tissue sections compared to 10 μm and 20 μm tissue sections used in protocols 2 and 3; it might be argued, therefore, that the reason protocol 4 extracted more RNA than protocols 2 and 3 was higher input of tumor tissue. However, protocol 1 had the highest input of tissue, 80 μm, yet extracted less RNA than the other protocols. Thus, the quantity of extracted RNA may or may not be related to tissue input. Protocol 2, which used only 10 μm of tissue input, may be a good choice when the amount of available tissue is scarce.
When we compared the results of the ten year old and recently archived months old samples in the extraction of biologically useful RNA (Tables 5 and 6), there were no significant differences in quality of extracted RNA between these different aged sample groups. The months old samples showed somewhat higher mean and median quantities of extracted RNA, except for Protocol 3, which extracted slightly higher amounts of RNA from the older tissues. All protocols, without exception, extracted biologically useful RNA in a higher percentage of attempts for months old compared to ten year old samples, although biologically useful RNA was still successfully extracted from each of the older samples a high percentage of the time.
Our RT-PCR data indicated that biologically useful RNA extracted from archivally stored FFPE tissue is suitable for RT-PCR analysis if the final product of amplification is designed to be 151 bp or less. We had a success rate of 100% for both the ten year old and more recently archived months old samples using primers for the housekeeping gene G6PD that amplified 67 bp and 151 bp. Successful amplification was achieved in none of our ten year old samples and in only 43% of our recently archived months old samples if the amplified fragment was 242 bp. This result is consistent with expected RNA degradation over time. However, the lower quantity of extracted RNA from ten year old samples that was used for the RT-PCR may also have contributed to the result. Liu et al., who cleverly designed the primers also used in this study, reported successful RT-PCR of 91% and 76% for 67 bp and 151 bp fragments of G6PD mRNA, respectively, but only 5% for 242 bp fragments measured from archival histologic specimens of diverse tissues (including decalcified specimen from bone) between one and 15 years old. RT-PCR of genes other than G6PD or use of RNA extracted from FFPE from other tissue sources could potentially produce different results.
All four protocols were time-consuming (one working-day for protocols 1, 3, and 4; two days for protocol 2 because of an overnight Proteinase K digestion), but easy to use. We personally appreciated the speed and quantity of RNA extracted using Protocol 4.