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RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathology for diagnosis. In the present study, we have set-up a method based on high performance liquid chromatography (HPLC) to investigate the effects of different fixatives on RNA. By the application of the presented method, which is based on the Nuclease S1 enzymatic digestion of RNA extracts followed by a HPLC analysis, it is possible to quantify the unmodified nucleotide monophosphates (NMPs) in the mixture and recognize their hydroxymethyl derivatives as well as other un-canonical RNA moieties. The results obtained from a set of mouse livers fixed/embedded with different protocols as well from a set of clinical samples aged 0 to 30 years-old show that alcohol-based fixatives do not induce chemical modification of the nucleic acid under ISO standard recommendations and confirm that pre-analytical conditions play a major role in RNA preservation.

This chapter provides a home-made method of obtaining RNA suitable for reverse transcription and PCR analyses from formalin-fixed and paraffin embedded (FFPE) specimens, even of autopsy origin. This protocol is based mainly on a deparaffinization step, a proteolytic digestion with Proteinase K, phenol purification, and alcohol precipitation, using a point-to-point protocol with notes and references to guide the researcher into the laboratory practice. A section of the chapter is specifically dedicated to RNA handling, because of the instability of this macromolecule. Specific precautions are suggested in order to prevent the degradation of RNA during the extraction and to avoid the accidental introduction of RNases from external sources.

The assessment of nucleic acid degradation levels is essential to guarantee the accuracy and reproducibility of the experimental results. This step becomes more important when working with fixed and paraffin-embedded (FPE) tissues, where the variable and unpredictable effects of the fixation processing on macromolecule preservation are dependent not only on the intrinsic properties of the fixative but also on the tissue-related preanalytical variables. In this chapter, several methods for the assessment of nucleic acid integrity are described, the focus being especially on their accuracy in FPE and suitability according to the availability and nature of the starting material. In particular, some PCR-based assays have also been reported as useful methods for integrity estimation when DNA/RNA starting material is very poor. Moreover, special attention has been given to RNA quality assessment, which commonly relies on methods based on the detection of ribosomal RNA as an indirect measure of target mRNA integrity. In this context, the limitations of these approaches in the quality assessment of RNA extracted from FPE have been pointed out, suggesting that PCR-based strategies, based on the detection of specific mRNA targets, still remain the best methods for the analysis of RNA isolated from archival material.

The extraction of useful RNA from FFPE tissues is often compromised for the extraction efficacy. Moreover, because it is resistant to extraction due to cross-linking with proteins, RNA in FFPE is not completely available for RT-PCR reactions. RNA, as DNA, is indeed modified in FFPE tissues by the presence of methylol addition. Prolonged fixations could favor further reactions with the above-mentioned groups, resulting in irreversible artifacts. The presence of the methylol group doesn’t allow the reverse transcription reaction, but the presence of the majority of these groups could be removed by simply heating the RNA extracts in formalin-free buffers. This chapter provides two simple temperature treatments to demodify RNA obtained from FFPE. In our experience the demodification treatment seems to improve the RNA recruitment only in old samples. The methods described hereafter could be added as routine procedure to the RNA extraction protocols especially for very old samples.

Several procedures are currently available for the quantification of DNA and RNA. However, not all these methods are equally useful for the accurate measurement of nucleic acid concentration extracted from fixed and paraffin-embedded samples. This is frequently due to the high degradation levels and low yields of extracted macromolecules, and to the presence of tissue contaminants that may or overestimate the quantity of DNA/RNA suitable for downstream analysis. The methods described in this chapter have been divided into two main categories, according to whether measurement is based on spectrophotometric measurement or on reading in the presence of a fluorescent dye. Each procedure has been illustrated referring to its advantages and/or limitations when used for quantification of nucleic acids extracted from archival samples. This section is focused especially on RNA measurement, for which the majority of the methods relies on detection of ribosomal RNA as an estimated measure of target mRNA quantity. A further section is dedicated to the absolute quantification of specific target sequences by means of the real-time-based amplification that results, particularly suitable when the starting material is extremely low.

Quantitative real-time PCR is a sensitive tool for quantitative expression analysis of RNA isolated from clinical samples and is increasingly being utilized in novel clinical diagnostic assays. It is an essential tool for the detection of degraded RNA, as that extracted from formalin-fixed paraffin-embedded (FFPE) tissues. Although it has unquestionable potential for expression analysis in archival case studies, there are still some biological and technical problems associated with its use, which can be partly overcome by a standardization of the protocols. The first part of this chapter provides a detailed description of the most commonly used real-time-based chemistries, SybrGreen and TaqMan, focusing especially on their advantages when applied in FFPE material. Specific suggestions have been included in order to improve the reliability of the expression results. The second part of the chapter is dedicated to the description of general guidelines that should be followed to optimize the design of quantitative real-time PCR assays. They have been adapted to RNA isolated from FFPE but should always be taken into account in any gene expression analysis to ensure its accuracy and reproducibility.

The presence of contaminating genomic DNA (gDNA) in RNA preparations is a frequent cause of false positives in RT-PCR-based assays aimed at gene expression analysis. Sometimes this phenomenon cannot be avoided even when specific precautions in the assay design are taken (e.g. design of intron-spanning primers), for example, when the target mRNA presents pseudogenes at the DNA level. For these reasons, the inclusion of a DNase digestion step is often necessary. In this chapter two alternative methods for RNA treatment with DNase are described. The choice of the most suitable method is largely dependent on the availability of starting RNA. The conventional DNase treatment requires a step of phenol/chloroform. This method is useful when an RNA solution is not pure and large RNA amounts are available, since this treatment causes about 50% RNA loss. The DNase digestion followed by enzyme heat inactivation is particularly suitable when an RNA starting quantity is very low because, theoretically, no further RNA is lost during heat treatment. This method may be very useful when an RNA has been extracted from small biopsies or cytologic specimens.