Dna molecular weight marker 8

Total RNA was extracted from HeLa cells using QIAzol Lysis Reagent (Qiagen SpA, Milan, Italy), and reverse transcription was performed according to Laforenza et al. [62 (link)]. cDNA amplification was performed by GoTaq^® Flexi DNA Polymerase (Promega, Milano, Italy), as previously described [62 (link)]. The primers used for amplification are listed in the Table 2. The PCR protocol consisted of an initial denaturation of 3 min at 95 °C followed by 35 cycles of denaturation at 96 °C for 30 s, annealing (see TM in Table 1) for 30 s, and extension at 72 °C for 30 s. Reverse transcription was always performed both either in the presence (positive) or in the absence (negative control) of reverse transcriptase enzyme. PCR products were separated on a 3% Nusieve^® (2:1) gel agarose, stained with ethidium bromide, and acquired with the Image Master VDS (GE Healthcare, Milano, Italy). The molecular weight of the PCR products was compared with the DNA molecular weight marker VIII (Roche Molecular Biochemicals, Monza, Italy).

Total RNA was isolated from fibroblasts using QIAzol Lysis Reagent (Qiagen SpA, Milan, Italy), and reverse transcription was performed as described in (Ferrera et al., 2021 (link); Negri et al., 2021 (link)). Reverse transcription was always performed in the presence (positive) or in the absence (negative control) of the reverse transcriptase enzyme (not shown), as shown elsewhere (Zuccolo et al., 2019 (link); Zuccolini et al., 2022 (link)). cDNA amplification was performed using KAPA SYBR FAST qPCR Master Mix (KAPA BIOSYSTEMS, United States), and the primers used for amplification are listed the Table 1. The conditions were as follows: initial denaturation at 95°C for 5 min; 40 cycles of denaturation at 95°C for 10 s; annealing and extension at 60°C for 30 s, PCR products were separated on a 3% Nusieve^® (2:1) gel agarose, stained with ethidium bromide, and acquired with the iBrightTM CL1000 Imaging System (Thermo Fisher Scientific Inc., United States). The molecular weight of the PCR products was compared with the DNA molecular weight marker VIII (Roche Molecular Biochemicals, Italy).

To detect E.g., Echinococcus multilocularis (E.m.) and other cestodes from extracted samples, a multiplex PCR assay was used as previously described by Trachsel et al. in 2007 [22 (link)] by using three pairs of primers listed in Table 2.
The PCR reaction mixture was carried out in a final volume of 25 µL including 12.5 µL QuantiTect Probe PCR Master Mix (Qiagen, Toronto, Canada) (1× final concentration), Milli-Q water RNAse-free (9 µL), 2.5 µL each forward and reverse primer (2 µM) and 1 µL DNA template. The amplification program consisted of an initial denaturation step of 15 min at 95 °C, followed by 40 cycles of 30 s at 94 °C, 90 sec annealing at 58 °C and 10 sec at 72 °C, and 1 cycle of 5 min at 72 °C. Amplicon products were electrophoretically separated in 2% agarose gel under standard conditions. DNA Molecular Weight Marker VIII (Roche, Basilea, Switzerland) was used for DNA sizing. The products were treated with nontoxic SYBR^® Green DNA Gel Stain (Invitrogen, Carlsbad, CA, USA), and visualised using standard UV transillumination.