**Protocol 2: Phenol-chloroform method (modified by our team)**

By following the same reasoning used to modify the RNA extraction protocol by Trizol method, the current study made the option to add a DNA purification step using the QIAamp DNA Blood Mini Kit commercial columns (Qiagen, Hilden, Germany) to check the impact from residual and potential contaminants.

As it was described in the previous protocol, the samples were transferred to the column commercial kit – QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany), after being in‐ cubated with 3M sodium acetate and absolute ethanol. Subsequently, they were centrifuged at 8,000 rpm for 1 min in the 5418- R microfuge (Eppendorf AG, Hamburg, Germany). The filtrate was discarded, and the column was transferred to a new tube and 500 µL of buffer AW1 were added to it. Then, the samples were centrifuged at 8,000 rpm for 1 min in the 5418-R microcentrifuge (Eppendorf AG, Hamburg, Germany).

After the filtrate was discarded and the column transferred to a new tube, a second wash was performed using 500 µL buffer AW2. The samples were centrifuged at 14,000 rpm for 3 min in the 5418-R microcentrifuge (Eppendorf AG, Hamburg, Germany).

Columns were transferred to a 1.5 mL DNases- and RNases-free microtube and 200 µL of buffer AE were added to the center of the column. The samples were then incubated at room temperature for 5 min, and it was followed by centrifugation at 8,000 rpm for 1 min in the 5418-R microfuge (Eppendorf AG, Hamburg, Germany). The samples were stored at –20°C until they were used.

#### **5.1. DNA quality quantification and analysis**

The DNA samples' concentration and purity were set by spectrophotometry in Nano‐ Drop®ND-2000 machine (Thermo Fisher Scientific, Wilmington, DE). Samples with absorb‐ ance ratios (A280 / A260nm) between 1.7 and 1.9 were considered to be appropriate.

#### **5.2. Results and comments on the herein described DNA extraction protocols**

Despite the DNA produced with suitable concentrations and purity degrees, not all the sam‐ ples obtained by the phenol-chloroform method showed amplification in real-time PCR re‐ actions. It corroborated the results by Witchell et al. (2008) [5], but it did not confirm the data obtained by Körbler et al. (2003) [8] and Antica et al. (2010) [19].

A significant improvement was observed in the quality of the samples and adequate ampli‐ fication in real-time PCR reactions performed according to method 2. Thus, DNA extraction from FFPE samples of the three tested protocols has become more viable. However, taking into account both the protocol practicality and the cost, the protocol 1 (Ambion commercial Kit) was used as the standard method for the current research.
