**4. RNA extraction from FFPE tissues**

**3. Preparing FFPE tissue sections for nucleic acids extraction**

embedded (FFPE) according to the standard methods described in the literature.

subsequently subjected to RNA and DNA extraction processes.

tion process started, as it is described in the following sections.

thesis primers and different primer sequences, among others [18].

same process was used in the DNA extraction performed by our team.

amplification in PCR reactions [17].

30 Nucleic Acids - From Basic Aspects to Laboratory Tools

PCR inhibitors.

All samples included in the studies conducted by our team result from biopsies performed in diffuse large B cell lymphoma patients, from lymph node samples or from reaction amyg‐ dala samples stored in the Pathology Department of Hospital das Clínicas, Medical School of University of São Paulo. All the herein used samples were formalin-fixed and paraffin-

Four 20-µm thick cuts were performed in each sample using routine histological techniques. The sections were placed into 1.5 mL RNase- and DNase-free microtubes, and they were

The first protocol used to prepare the cuts in the nucleic acid extraction process (suggested for the majority of commercial kits and protocols) consisted of deparaffinizing the sections with xylene and of rehydrating them with ethanol. In order to do so, 1 mL xylene PA was added to each sample (Synth® Diadema, SP, Brazil), and it was followed by homogenization using Vortex Genie 2T (Scientific Industries, Inc., Bohemia, NY, USA) and by incubation at 50°C, for 5 min, in digital thermomixer (Eppendorf AG, Hamburg, Germany). After incuba‐ tion, the samples were centrifuged at maximum speed for 5 min in the R-5418 microfuge (Eppendorf AG, Hamburg, Germany). The xylene was discarded and the cell button was washed two times with absolute ethanol (Merck KGaA, Darmstadt, Germany). The superna‐ tant was discarded after each wash. After the cell button was completely dried, the extrac‐

Moreover, even after some RNA extraction methods that allowed finding some viable op‐ tions were compared, it was observed that not all the extracted samples showed successful

None of the previous studies available in the literature described the possible factors that could influence the amplification success. Therefore, the current study made the option of investigating some of the potential interferences in the nucleic acid obtainment process, namely: tissue fragment size, blocks' storage time, used fixative type, different cDNA syn‐

After the aforementioned study, the tissue preparation protocol for RNA extraction process was modified by including a washing step. It consisted of using 1 ml phosphate buffer sal‐ ine (PBS) with 5 min incubation at room temperature, followed by full speed centrifugation for 5 min in the R-5418 microfuge (Eppendorf AG, Hamburg, Germany). This procedure is done to remove possible fixative residues that could work as PCR inhibitors [18]. Next, the

Such protocol change was done under the assumption that the amplification failure in PCR reactions could be caused by the presence of contaminants such as fixative waste working as

The results show that the PBS washing step inclusion in the samples' extraction preparation process led to some statistically significant advantages such as the obtainment of better Three different RNA extraction protocols were tested, as described below:

### **Protocol 1: Commercial kit RECOVERALL Total Nucleic Acid Isolation Optimized for FFPE Samples (Ambion, Inc., Austin, Texas, USA)** [17–18]

Two hundred (200µ l) of digestion buffer and 5 µL protease were used for tissue lysis in each sample. It was followed by incubation for 15 min at 50°C and for 15 min at 80°C. Next, RNA isolation was held by the addition of 790 µL buffer containing absolute ethanol and by the subsequent passage through the separation column. After washing the column with two wash buffers, realized treatment with DNAse and further washing with two buffers, accord‐ ing to the manufacturer's instructions. The eluted RNA was obtained by using 60 µL of the kit elution buffer at RT. After incubation for 5 min at room temperature, the samples were centrifuged at maximum speed and the obtained RNA was stored at –80°C until its use.

### **Protocol 2: Paradise®Whole Transcript RT Reagent kit System (Arcturus Bioscience, Inc., Mountain View, California, USA)** [17]

Incubation with buffer containing proteinase K was held for 20 h, at 37°C, after the samples' preparation process, to digest the proteins. The RNA isolation was performed by two suc‐ cessive washes using buffer containing the ethanol kit. Then, the samples were purified by passing them through the column kit according to the manufacturer's instructions. Subse‐ quently, samples were incubated with buffer containing DNase at 37°C for 15 min and at 4°C for 1 min. DNase inactivation was performed by incubating the samples at 70°C for 10 min and at 4°C for 1 min. The RNA samples were stored at –80°C until they were used.

## **Protocol 3: Trizol extraction method (Invitrogen, UK)** [17]

The RNA extraction by Trizol method was performed as recommended by Körbler et al. (2003) [8] and Antica et al. (2010) [19]. The tissue was digested by incubating the samples in buffer containing 10 mM NaCl, 500 mM Tris pH 7.6, 20 mM ethylenediaminetetraacetic acid (EDTA), 1% sodium dodecyl sulfate (SDS) and 500 µg/mL proteinase K. First, the sample was incubated at 55°C, for 3 h; then, it was incubated at 45°C overnight with proteinase K inactivation by elevating the temperature to 100°C for 7 min in the next day. Finally, all sam‐ ples were subjected to RNA extraction process according to the classic Trizol method previ‐ ously described by Chomczynski and Sacchi (1988) [20]. The obtained RNA was stored at – 80°C until it was used.

#### **4.1. Evaluating RNA concentration and quality**

NanoDrop equipment (NanoDrop Technologies, Inc. Wilmington, DE) was used to evaluate the concentration and purity of RNA samples extracted according to the three protocols de‐ scribed above. RNA amounts above 50 ng/µl with purity between 1.7 and 1.9 were consid‐ ered to be suitable.

### **4.2. Results and comments on the herein described RNA extraction protocols**

The purity levels and degrees obtained by the three protocols were satisfactory (over 50 ng/µL and purity concentrations between 1.8 and 1.9). However, only samples obtained ac‐ cording to protocols 1 (Ambion) and 2 (Arcturus Bioscience) showed appropriate amplifica‐ tion in real-time PCR reactions [17].

Despite the RNA produced with appropriate concentrations and purity degrees, the samples obtained by the Trizol method showed no amplification in real-time PCR reactions. It cor‐ roborates the results found by Witchell et al. (2008) [5], but it did not confirm the data ob‐ tained by Körbler et al. (2003) [8] and Antica et al. (2010) [20].

After these results were published, it was decided to check the impact of the residuals and potential contaminants. Thus, the purification step using alcohol in a porous column of the QIAamp®Viral RNA Mini Kit for commercial extraction (Qiagen) was adopted. In brief, af‐ ter isopropyl alcohol was added to the samples, DNA was filtered using the purification col‐ umn, according to the manufacturer's recommendations. It was done by transferring the samples to the Kit's purification columns, which were centrifuged at 8,000 g for 1 min. Sub‐ sequently, the column products were moved to other tubes. Next, 500µ L buffer AW1 was added to column and it was once again centrifuged at 8,000 g for 1 min. After the filtrate was discarded and the column transferred from the column to another tube, 500 µL Buffer AW2 was added to the solution and a new centrifugation was performed at 14,000 g for 3 min. Then, the column was shifted into a sterile 1.5 mL tube and 60µ L elution buffer was added to it. After the reaction was incubated for 5 min at RT, the tubes were centrifuged at 8,000 g for 1 min and the obtained RNA was stored at –80°C.

A significant improvement was found in the quality of the samples as well as in their ade‐ quate amplification in real-time PCR reactions. Thus, RNA extraction from FFPE samples of the three tested protocols became feasible. However, protocol 1 (Ambion commercial Kit) was used as the standard method in current research by taking into account the protocol's practicality and cost.
