**2.1.1 Whole blood**

Peripheral blood remains the most commonly studied tissue due to the minimally invasive nature of sample collection and the vascularization of most tissues. Peripheral whole blood is a rich source of validated and potential biomarkers, whether they are protein, genomic, or metabolic in nature. While the methods for extraction and profiling of blood DNA are well established, the isolation of RNA and microRNA from whole blood, and studies on their transcript abundance (commonly called gene expression studies), still pose many technical challenges. These include transcriptomic changes induced by *ex vivo* handling and the interference of highly abundant globin mRNA.

Pre-analytical variables such as the degradation of RNA by endogenous RNases and unintentional expression of individual genes after drawing blood could lead to false assessment of potential markers. The introduction of blood collection systems containing stabilizing additives has significantly improved the RNA quantity and quality of blood samples (Rainen et al., 2002; Thach, 2003). RNA stabilization systems have the advantage of storing the collected samples at more accessible temperatures before shipment to the laboratory for analysis, resulting in reduced pre-analytical variability. A well-described method for RNA stabilization in human blood is the PAXgeneTM system (Chai et al., 2005; Rainen et al., 2002). The Tempus™ Whole Blood RNA isolation system offers an alternative approach to peripheral blood RNA isolation suitable for gene expression profiling as well (Asare et al., 2008). Recently RNAlaterTM, a common stabilization reagent for RNA in cells and tissues, has been successfully used for RNA stabilization in human peripheral blood (Weber et al., 2010). The downside of the latter method is that pre-filled RNAlaterTM blood collection tubes are not currently available commercially.

All the described methods are able to stabilize transcription and isolate total RNA with good quality and in appropriate quantities. However, RNA stabilization/isolation methods can critically impact differential expression results. For example, the failure of PAXgeneTM to stabilize specific transcripts was reported in several studies (Asare et al., 2008; Kågedal et al., 2005). Until more broad studies are done, it is recommended that a researcher should prevalidate the whole blood stabilization/isolation conditions with the transcripts of interest. We find that strict adherence to the manufacturer's protocol for collection and storage, including how the reagent is mixed with the blood at the time of collection, is critical to successful expression profiling.

The discovery of microRNAs has opened new opportunities for markers in the diagnosis of cancer (Wang et al., 2009). MicroRNAs are small (typically ~22 nt in size) regulatory RNA molecules that function to modulate the activity of specific mRNA targets and play important roles in a wide range of physiologic and pathologic processes (Mattick & Makunin, 2005). MicroRNAs are an ideal class of blood-based biomarkers for disease detection because: (i) miRNA expression is frequently dysregulated in disease, (ii) expression patterns of miRNAs are tissue-specific, and (iii) miRNAs have unusually high stability in most tissues and can be recovered from formalin-fixed, paraffin embedded samples.

Novel Tissue Types for the Development of Genomic Biomarkers 275

plasma or serum samples (< 1 mL) usually falls below the limit of accurate quantification by spectrometry and calls for an alternative way to assess the efficiency of nucleic acids recovery. Several serum/plasma extraction kits are now available commercially through Qiagen, Norgen and other companies. These kits successfully address the problems mentioned above, employing column-based purification methods and various carriers. We suggest the use of carefully selected extraction spike-ins to allow researchers to evaluate the

Circulating tumor cells (CTCs) are cells that have been sloughed off of primary tumors and circulate in the bloodstream. Their numbers can be very small (1-10 cells per mL of whole blood) and these cells are not easily detected. Even though CTCs were first observed by Thomas Ashworth back in 1869, the technology with the requisite sensitivity and reproducibility to detect CTC in patients with metastatic disease was developed only recently (Sleijfer et al., 2007). While the presence of circulating tumor cells themselves can serve as a marker of poor clinical outcome, there is an opportunity to develop new biomarkers by studying the gene or protein expression in these cells. Changes in the phenotype of tumor cells can occur after the original diagnosis and resistance to a treatment can only be inferred after the treatment has failed. CTCs offer a tool to understand the

Recently, CTCs have been the target of multiple molecular profiling studies (Bosma et al., 2002; Punnoose et al., 2010; Smirnov et al., 2005; Tewes et al., 2009). mRNA expression and DNA mutations can be measured from captured CTCs. RT-PCR using a multi-marker panel of cancer-associated genes was found to be the most sensitive technique for the detection of CTC in blood of breast cancer patients (Bosma et al., 2002; Tewes et al., 2009). Another approach involves the analysis of CTC-enriched samples by microarray gene expression profiling, where numerous genes like S100A14 and S100A16 have been detected (Smirnov et

The method of collecting capillary blood on filter paper was introduced in Scotland by Robert Guthrie in 1963 and since then has become a mainstream approach for blood sample collection from newborns in more than 20 countries (Consultant Paediatricians and Medical Officers of Health of the SE Scotland Hospital Region, 1968; Scriver, 1998). These samples were found invaluable for screening for congenital metabolic disorders. Dried blood spots (DBS) are easily acquired through a simple needle stick and transfer to paper cards that are stored and handled at room temperature in ambient atmospheric conditions. This approach eliminates many costly, time-consuming, and unpleasant aspects of sample collection, and can also significantly reduce the cost for shipping samples. The collection of DBS samples requires very little infrastructure and can be done in resource-limiting locations. Vidal-Taboada and colleagues even showed that both patients and investigators prefer this as a

The limitation of small sample volume has restricted the usage of dried blood spots for the development of molecular diagnostics until recently. Advances in technology have

complex biology of tumor cells, without the need of invasive biopsies.

method of DNA collection and storage (Vidal-Taboada et al., 2006).

efficiency of the circulating nucleic acids isolation.

**2.1.3 Circulating tumor cells** 

al., 2005).

**2.1.4 Dried blood spots** 

Several studies have reported optimized isolation protocols to enhance the recovery of microRNAs in the stabilized samples. For example it was shown that microRNAs could be isolated from PAXgene-stabilized blood of sufficient quantity and quality that is suitable for downstream applications (Kruhøffer et al., 2007).

Another problem hampering the analysis of microarray gene expression data in whole blood is the presence of globin. Globin mRNA in red blood cells accounts for over 70% of all mRNA in whole blood and interferes with the accurate assessment of other genes (Field et al., 2007; Wright et al., 2008). Several approaches have been developed to mitigate this effect and tested in microarray experiments (Liu et al., 2006; Vartanian et al., 2009; Wright et al., 2008). Globin reduction techniques based on biotinylated DNA capture oligos (Ambion GLOBINclear processing protocol) produced sensitive results but was least reproducible among all the methods tested (Vartanian et al., 2009). An alternative protocol with globin PNAs (peptide nucleic acid inhibitory oligos) proved to be the best in sensitivity and reproducibility, but was the most time-consuming and required the highest amount of total RNA input (Liu et al., 2006; Vartanian et al., 2009). An alternative approach was suggested by Eklund and colleagues (Eklund et al., 2006). NuGEN's Ovation WB sample preparation protocol, based on single primer isothermal amplification (SPIA), generates cDNA target. The hybridization kinetics of the cDNA target are less affected than cRNA targets by the abundant globin RNA present in whole blood extract. The high specificity and sensitivity of cDNA targets, and the highly reproducible SPIA protocol have been shown to be as good or better for mitigating the interference of globin transcripts compared to other protocols (Fricano et al., 2011; Li et al., 2008; Parrish et al., 2010). The strong performance of this technique, and the relatively low input requirements (50ng of total RNA) have made the NuGEN Ovation WB protocol the method of choice for gene expression profiling in the microarray community.

#### **2.1.2 Serum and plasma**

Both plasma and serum are widely used specimen types for molecular diagnostics. Nucleic acids that can be found in small amounts in cell-free preparations of whole blood are frequently called "circulating nucleic acids". To date, a number of studies show that plasma and serum nucleic acids can serve as both tumor- and fetal-specific markers for cancer detection and prenatal diagnosis, respectively. For example, several studies reported increased concentrations of DNA in the plasma or serum of cancer patients sharing some characteristics with DNA of tumor cells (Leon et al., 1977; Stroun et al., 1989). Interestingly, DNA levels decreased by up to 90% after radiotherapy, while persistently high or increasing DNA concentrations were associated with a lack of response to treatment (Anker et al., 2001). RNA has also been found circulating in the plasma or serum of normal subjects and cancer patients (Feng et al., 2008; Tsui et al., 2002, 2006). The recent discovery that serum and plasma contain a large amount of stable miRNAs derived from various tissues/organs has lead to multiple studies on circulating miRNA expression as well (Mitchell et al., 2008; Chen et al., 2008; Zhu et al., 2009).

Analysis of circulating nucleic acids, however, requires modified extraction methods to utilize plasma or serum as the source material. First, plasma and serum are biospecimens that have a very high concentration of protein that can interfere with sample preparation and detection techniques. Second, the yield of circulating nucleic acids from small volume plasma or serum samples (< 1 mL) usually falls below the limit of accurate quantification by spectrometry and calls for an alternative way to assess the efficiency of nucleic acids recovery. Several serum/plasma extraction kits are now available commercially through Qiagen, Norgen and other companies. These kits successfully address the problems mentioned above, employing column-based purification methods and various carriers. We suggest the use of carefully selected extraction spike-ins to allow researchers to evaluate the efficiency of the circulating nucleic acids isolation.
