**2.4.2 Skin tissue alternatives**

Synthetic skin is a relatively new surrogate tissue that lends itself to investigation of a wide variety of processes while reducing the need for volunteer recruitment or laboratory animal testing (Poumay & Coquette, 2007). For extracting nucleic acids, we have found that synthetic skin is less susceptible to nucleic acid degradation and more easily homogenized than real human skin (data not shown). Synthetic skin has recently been used to study processes such as wound healing (Koria et al., 2003), epithelial development (Taylor et al., 2009), effects of cosmetics on skin (Faller et al., 2002), and even differential gene expression in skin disorders.

Novel Tissue Types for the Development of Genomic Biomarkers 279

can identify genetic aberrations in the genomes of these cells and understand or diagnose, non-invasively, the pathology of the patient's disease. However, the extraction and purification of nucleic acids in feces is quite challenging due to its low abundance and the high level of contaminants like humic acid. Thankfully, there are a number of commercial kits available for fecal DNA isolation, and new techniques such as synchronous coefficient of drag alteration (SCODA) show promise in further purifying and concentrating this rare DNA (Broemeling et al., 2008; Marziali et al., 2005). Interestingly, both the amount and integrity of DNA in feces have been shown to identify colorectal cancer patients (Klaassen et al., 2003; Osborn & Ahlquist, 2005). A variety of mutations found in this DNA have been identified in the stool of colorectal cancer patients. Genes identified with mutations include KRAS, TP53 and APC, among several others (Osborn & Ahlquist, 2005; Young & Bosch, 2011). The most interesting of these is the adenomatous polyposis coli gene (APC). Mutations in the APC gene have been shown to drive the growth of adenomas, and their identification in stool samples allows the early detection of early stage colorectal neoplasia (Jen et al., 1994; Traverso et al., 2002). Analysis of fecal DNA has also been used to identify

The isolation and analysis of RNA from fecal samples has also gained a great deal of attention. While less stable than DNA, RNA provides a snapshot of the transcriptional activity of exfoliated cells; reflecting both genomic and environmental influences. Changes in gene expression may more fully reflect a target tissue's response to therapeutic agents. Alexander and Raicht demonstrated the ability to extract RNA from stool and suggested its use as a method for the early detection of colon tumors (Alexander & Raicht, 1998). One such transcript with potentially diagnostic value is cyclooxygenase 2 (COX-2) which can separate colorectal cancer patients from healthy patients (Kanaoka et al., 2004). Still others are exploiting fecal RNA to better understand infant health (Chapkin et al., 2010; Davidson

Urine is an ideal source for the identification of new biomarkers as it is easily and noninvasively collected. It has long been a standard fluid for the measurement of metabolites, proteins, and infectious agents. Recent data has demonstrated that not only can these traditional analytes can be identified, but RNA, DNA miRNA can be extracted and profiled. While less stable than the other nucleic acids, mRNA can be detected in urine. Keller and colleagues have demonstrated that this stability is likely due to protection of the mRNA in protein/lipid vesicles called exosomes (Keller et al., 2011; Nilsson et al., 2009). Further, mRNA patterns from urine sediments have been suggested for the development of ovulation and fertility biomarkers (Campbell & Rockett, 2006). miRNAs have also been uniquely identified in urine (Weber et al., 2010), and their stability has also been linked to exosomes (Record et al., 2011; Valadi et al., 2007). Differential detection of miRNAs in urine is showing promise in the non-invasive detection of lupus, nephropathy, renal allograft rejection and urothelial cancer (Lorenzen et al., 2011; Wang et al., 2010; Wang et al., 2011;

Urinary DNA is a complex target, with both host and non-host DNA being present and clinically relevant. Patient DNA is readily extracted from urine with methylation patterns that have been shown to have utility in the diagnosis of cancer and kidney injury (Chen et

pancreatic adenocarcinoma (Caldas et al., 1994).

et al., 1995; Kaeffer et al., 2007).

**2.7 Urine** 

Yamada et al., 2011).

Yao et al. identified the overexpression of type I IFN-inducible genes in psoriatic biopsies by comparing biopsies of normal, healthy donor skin and non-lesional skin to psoriatic donor skin (Yao et al., 2008). To better understand the degree of type I IFN-inducible gene overexpression in psoriasis, blood from healthy donors and normal keratinocyctes (EpiDerm, MatTek, Inc.) were stimulated with various members of the type I IFN family. Ex vivo blood and in-vitro keratinocyte data showed overall agreement in up-regulated type I IFN-inducible genes. While only 1% of upregulated probes from the stimulation study were overexpressed in non-lesional compared to normal skin, 11.7% of the upregulated probes were overexpressed in lesional compared to non-lesional skin, suggesting type I IFNs may be a prospective target for psoriatic treatment.

### **2.5 Hair follicles**

Hair follicles are different from skin and blood, in that they are made up of stem cells, which control the growth and cycling of hair. The stem cells are contained within the follicle and are often called the bulge. It is this fact which makes hair follicle gene expression particularly intriguing: "stem cells in the epidermis and hair follicle serve as the ultimate source of cells for both of these tissues, understanding the control of their proliferation and differentiation is key to understanding disorders related to disruption in these processes," (Cotsarelis, 2006).

Advances in hair follicle extraction, isolation, and amplification techniques along with the relative ease of collection of the tissue, and the abundance on most, hair follicle collection is being increasingly examined as a good investigatory and clinical biomarker tissue. To date most research has been in diseases involving skin conditions (Ohyama et al., 2006). However, hair follicles are also being examined for markers in to quantify exposures to pharmaceuticals (Reiter et al., 2008) or toxicology to certain drug targets (Kim et al., 2006).

Hair follicles are obtained using tweezers, grasping at the hair as near to the scalp as possible, and quickly yanking upwards. The follicle should be clearly present and immediately preserved in the appropriate preservation solution. For those with longer hair, we have found it helpful to cut the hair close to the follicle, before preservation. Although it is possible to achieve results with a single or a few (3 follicles), it is often better to acquire a larger set (15 follicles), to ensure the needed mass for evaluation will be met. The follicles for the experiment should be taken from a similar location for each extraction, as there might be slight gene expression changes with different hair locations (head, arm, and eyebrow). We recommend behind the ear for collection of the desired hairs for most applications. There are several different preservation solutions such as RNAlater (Ambion) or SD Lysis Buffer (Promega). Following preservation, follow the manufacturer guidelines on storage and extraction/isolation of the RNA.

#### **2.6 Feces**

Often overlooked, stool is an important source of potential biomarkers for a number of clinical indications. While the identification of infection and various metabolic imbalances are easily identified, feces can also yield RNA, DNA and miRNA for use in biomarker development. This is largely due to the shedding of epithelial cells in the gastrointestinal track (Osborn & Ahlquist, 2005). With the use of highly sensitive detection techniques, one can identify genetic aberrations in the genomes of these cells and understand or diagnose, non-invasively, the pathology of the patient's disease. However, the extraction and purification of nucleic acids in feces is quite challenging due to its low abundance and the high level of contaminants like humic acid. Thankfully, there are a number of commercial kits available for fecal DNA isolation, and new techniques such as synchronous coefficient of drag alteration (SCODA) show promise in further purifying and concentrating this rare DNA (Broemeling et al., 2008; Marziali et al., 2005). Interestingly, both the amount and integrity of DNA in feces have been shown to identify colorectal cancer patients (Klaassen et al., 2003; Osborn & Ahlquist, 2005). A variety of mutations found in this DNA have been identified in the stool of colorectal cancer patients. Genes identified with mutations include KRAS, TP53 and APC, among several others (Osborn & Ahlquist, 2005; Young & Bosch, 2011). The most interesting of these is the adenomatous polyposis coli gene (APC). Mutations in the APC gene have been shown to drive the growth of adenomas, and their identification in stool samples allows the early detection of early stage colorectal neoplasia (Jen et al., 1994; Traverso et al., 2002). Analysis of fecal DNA has also been used to identify pancreatic adenocarcinoma (Caldas et al., 1994).

The isolation and analysis of RNA from fecal samples has also gained a great deal of attention. While less stable than DNA, RNA provides a snapshot of the transcriptional activity of exfoliated cells; reflecting both genomic and environmental influences. Changes in gene expression may more fully reflect a target tissue's response to therapeutic agents. Alexander and Raicht demonstrated the ability to extract RNA from stool and suggested its use as a method for the early detection of colon tumors (Alexander & Raicht, 1998). One such transcript with potentially diagnostic value is cyclooxygenase 2 (COX-2) which can separate colorectal cancer patients from healthy patients (Kanaoka et al., 2004). Still others are exploiting fecal RNA to better understand infant health (Chapkin et al., 2010; Davidson et al., 1995; Kaeffer et al., 2007).
