Fresh tissue will usually provide high quality DNA since it is not subjected to the crosslinking formalin causes, however, nucleic acid must be isolated from it quickly, or the tissue needs to be preserved. Typically, to store fresh tissue it is frozen in either a cutting media such as optimum cutting temperature (OTC) compound or snap frozen. OTC allows for the cutting of frozen section slides, which can be used for histological analysis and for nucleic acid purification. Freezing fresh tissue allows for the long-term storage of high quality nucleic acid. Fresh tissue is typically taken at the time of surgery, requiring protocols in place to quickly select and freeze the tissue. Due to the need for clinical pathologic analysis, the amount of tissue that can be taken for molecular analysis is often limited, especially if not for clinical testing. This tissue is usually selected by gross examination only, which can lead to missing the desired tissue. Therefore, it is recommended to always confirm the type of tissue through histologic analysis.

The typical workflow for any pathology department involves receiving specimens, performing a gross examination/description, and then preparing the specimen for histologic analysis., as described in Chap. 2. As formalin fixation and paraffin embedding of tissue is a standard procedure, there is a wealth of archived material in almost all pathology departments. FFPE tissue has several advantages over fresh frozen tissue that make it so widely used. These include being able to store blocks at room temperature and proving better histology. Formalin fixes tissue by forming cross-links between proteins, DNA, and RNA. Unfortunately, the purification of nucleic acid from FFPE material causes shearing of the nucleic acid. This is a major drawback and can limit the ability to use FFPE material in molecular analysis, depending on how long of nucleic acid fragments are needed for testing. Figure 4.2a shows an agarose gel of DNA purified from both fresh frozen and FFPE tissue. The large smears in the FFPE columns show sheared DNA of varying lengths, when in comparison, the fresh frozen column has a large band of DNA at the top of the gel indicating large-sized DNA. Figure 4.2b shows an RNA electropherogram showing the size of RNA fragments from fresh and FFPE tissue. The FFPE RNA sample is predominately composed of small fragments, when in contrast, in the fresh sample, one can see large 18S and 28S ribosomal RNA (rRNA) peaks. Despite these issues, FFPE tissue can often be used for a variety of testing [8].

There are several other fixatives apart from formalin that can be sometimes used in pathology departments. Alcohol fixation (with either ethanol or methanol) will provide higher quality DNA and RNA than formalin. Bouin’s fixative or solution is a combination of formalin, picric acid, and acetic acid. While it provides excellent histology, the acid component degrades nucleic acid making most Bouin’s-fixed tissue unusable for molecular analysis. The same is true for many of the acid-based decalcification solutions used in pathology [9].

Quantitative real-time polymerase chain reaction (qPCR) utilizes a traditional PCR reaction with the addition of a reporter probe (Fig. 4.3) [12]. The reporter probe has both a fluorescent reporter and a quencher with a sequence of complimentary nucleic acid for binding. Due to the close proximity of the reporter and quencher, no fluorescence is seen. As the PCR primers get extended and approach the reporter probe, the 5′–3′ exonuclease activity of the Taq polymerase degrades the bound probe, releasing the fluorescent marker. The fluorescent marker can then be detected since it is no longer in close proximity to the quencher. Like the name of the technique suggests, qPCR can be used to quantify the amount of DNA. The more DNA is present, the more reporter probe is bound and thus more fluorescence is detected once the marker is released. A variation of this technique is reverse transcription (RT) qPCR. This method can be used to quantify the amount of an RNA transcript. For this, messenger RNA is purified and undergoes a RT step to form complementary DNA (cDNA) before the qPCR is performed. One example of a typical use of this technique is quantifying the amount of cDNA to determine titers of nonhuman RNA/DNA (e.g., viral) within a human sample [13]. The results can either be compared to a set of standards to determine the absolute amount of the pathogenic DNA or more commonly when analyzing human DNA and with RTqPCR, the DNA or mRNA amount is compared to an internal standard. In this way, the results can be reported as a ratio of the specific nucleic acid to the standard. These results can then easily be compared with other samples. For expression analysis, this internal standard is usually a common (or housekeeper) gene with a known, relatively constant expression. In addition to the above-mentioned uses, qPCR can also be used for the detection of a PCR product similar to standard PCR except the analyte is detected in “real time” so a detection method such as running the DNA out on a gel is not needed.

Mutation-specific PCR uses primers in the PCR reaction that are specific for a certain single nucleotide pleomorphism (SNP), or variant. In this reaction, the primer is designed so that it will only extend if the nucleotide being interrogated matches the primer (Fig. 4.4). There are multiple ways in which the primer can be designed to achieve this, however, one of the most common is to make the 3′ most nucleotide the SNP. Even if all of the other nucleotides match and the primer binds, the primer cannot get extended by the polymerase. A variant of this procedure intentionally designs a mismatch next to the nucleotide being interrogated. Therefore, if the interrogated nucleotide also does not match it will create two mismatches next to each other, the probe cannot stay bound, and amplification will not occur. In this technique, when designing the primer, the base of interrogation is placed in position-1 from the 3′ base and the intentional mismatch is placed at-2 from the 3′ base. Mutation-specific PCR is a fast, sensitive way to quickly determine if a specific mutation is present. The downside is that the alteration being interrogated must be known in advance. Due to the amplification of the target, mutation-specific PCR can be a very sensitive means of detecting rare sequence variations. However, as with any PCR reaction the utmost care needs to be taken to avoid contamination.

In situ hybridization (ISH), introduced in Chap. 2, is a technique that can be used to detect RNA expression or DNA copy number. There are many variations of ISH depending on what is being detected, the type of probe, and the detection method. In general, it uses a nucleic acid probe to detect DNA or RNA in situ. This is often performed on touch preps, frozen section slides, or FFPE material. The probes are designed to be complimentary to the target sequence and are often labeled with a fluorescent (i.e., FISH) or chromogenic (i.e., CISH) reporter. A signal amplification step may be utilized for increased detection. DNA rearrangements and amplifications are typically detected with FISH (Fig. 4.5a) while RNA is often detected by CISH (Fig. 4.5b). For detection and quantification of the amount of a specific DNA or RNA, the probe is bound and the number of markers are counted. In the clinical setting, CISH is often used to detect Epstein-Barr virus-encoded RNA (EBER) inside human tissue [14]. One of the major advantages of ISH is the fact that it can be performed on a normal tissue slide allowing correlation with histology and confirmation that the proper cells are being interrogated. One of the drawbacks of ISH, similar to PCR, is that sequences near the target must be known. In addition, ISH is somewhat less sensitive as compared to PCR-based tests.