**5. DNA extraction from FFPE tissues**

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.,**

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.

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 –

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‐

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‐

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‐

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

tained by Körbler et al. (2003) [8] and Antica et al. (2010) [20].

**Mountain View, California, USA)** [17]

32 Nucleic Acids - From Basic Aspects to Laboratory Tools

80°C until it was used.

ered to be suitable.

tion in real-time PCR reactions [17].

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

**4.1. Evaluating RNA concentration and quality**

Two different RNA extraction protocols were tested (unpublished data) as described below.

### **Protocol 1: Phenol-chloroform method** [21]

After the sections were prepared and the button cell was completely dried as described in Section 3, tissue digestion was performed by adding 480 µL Tris-EDTA buffer (TE) and 20 µL proteinase K (200 mg/ml) to each sample. Next, these samples were incubated at 37°C for approximately 16 h in the digital thermomixer (Eppendorf AG, Hamburg, Germany). The temperature was then raised up to 90°C for 10 min to inactivate the proteinase K.

One (1) ml of the mixture was added to phenol:chloroform:isoamyl alcohol (Invitrogen Cor‐ poration, Carlsbad, CA, USA) at the ratio 25:24:1, respectively. After homogenization using the Vortex Genie 2T (Scientific Industries, Inc., Bohemia, NY, USA), samples were centri‐ fuged at 13,000 g for 15 min at the 5418-R microcentrifuge (Eppendorf AG, Hamburg, Ger‐ many). The supernatant was transferred to a fresh 1.5 ml DNase- and RNase-free microfuge tube, and 1 mL of the phenol:chloroform:isoamyl alcohol (25:24:1) mixture was added to it. Then, the centrifugation process was repeated under the same conditions.

The supernatant was transferred to a new 1.5 mL DNase- and RNase-free microtube and 20 µL of 3M sodium acetate and 900 µL of absolute ethanol (Merck KGaA, Darmstadt, Germa‐ ny) were added to it. The samples were homogenized by inversion and incubated at 20°C for at least 30 min. The samples were centrifuged at 13,000 g for 15 min. The supernatant was discarded and the pellet was completely dried. Samples were resuspended in 50 mL TE buffer and stored at –20°C, until they were used.
