Proflex pcr system thermal cycler

Two different templates were used for in vitro transcription (IVT): (I) the native SARS-CoV-2 Spike gene (Addgene 145670), and (II) the codon-optimized, GC-enriched spike-encoding construct from Pfizer-BioNTech ^{36 ,37} . Both genes were expressed from bacterial expression plasmids downstream of T7 promoters. Plasmids were isolated from E. coli cultures using the QIAprep Spin Miniprep Kit™ and subjected to single-enzyme digest with SnaBI (viral template) or Mlul (vaccine template). Linearized plasmid was then cleaned up by PCA and verified by Qubit Fluorometer™ and gel electrophoresis before IVT. Reactions were performed in parallel at escalating temperatures (30, 37, 42 C) using the Hi Scribe® T7 High Yield RNA Synthesis Kit (NEB) and the ProFlex PCR System Thermal Cycler (Applied Biosystems). Starting from 200 ng of template, reactions were allowed to proceed for two hours before TURBO™ DNase treatment. Transcription products were purified by PCA for sequencing. Notably, while IVT from the vaccine template was less efficient and had lower yields, vaccine transcripts were more resistant to RNaseIII fragmentation during library preparation.

Potential molecular determinants of resistance in the 16S rRNA gene were evaluated by Sanger sequencing. Briefly, DNA from four isolates with no gentamicin resistance (MIC ≤ 2 µg/mL) and five isolates with MIC ≥ 16 µg/mL was extracted by a freeze–thaw cycle and used for the PCR amplification of the complete sequence of the 16S rRNA gene (~ 1500 bp). Each PCR reaction was performed using the DreamTaq DNA polymerase kit (Thermo Scientific) with 20 pmol of primer 27F (5ʹ-AGAGTTTGATCMTGGCTCAG-3ʹ), 20 pmol of primer 1492R (5ʹ-GGTTACCTTGTTACGACTT-3ʹ) and 1 µL of DNA in a final volume of 50 µL. The reaction mixture was subjected to a protocol of amplification in a ProFlex PCR System thermal cycler (Applied Biosystems) that consisted of an initial step of 95 °C for 5 min followed by 35 cycles of 95 °C for 30 s, 57 °C for 30 s and 72 °C for 1 min, with a final extension step of 72 °C for 10 min.
The resulting amplicons were purified with the NucleoSpin Gel and PCR Clean-up kit (Macherey–Nagel) and sequenced in both directions employing the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) in an ABI 3500 automated capillary sequencer (Applied Biosystems). Forward and reverse sequences were assembled into contigs and aligned together with the ClustalW tool in MEGA software version 11 [13 (link)].

Quantitative PCR was performed, as we previously published [20 (link)]. Briefly, RNA was extracted from the mesothelium and mesothelioma samples using the High Pure FFPET RNA Isolation Kit (Roche). Concentration of the extracted RNA was measured with NanoDrop ND-1000 Spectophotometer (Nano DropTechnologies, Thermo Fisher Scientific, Waltham, MA). Reverse transcription was performed with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Thermo Fisher Scientific) in the ProFlex PCR System Thermal Cycler (Applied Biosystems, Foster City, CA, USA). Gene expression was analyzed using the Cobas z 480 instrument (Roche) according to the manufacturer’s instructions. Relative expression of selected genes was normalized against the endogenous control, RPLP0. The following gene expression assays were obtained from the Thermo Fisher Scientific: POU5F1 (Hs00999632_g1), NANOG (Hs04260366_g1), SOX2 (Hs01053049_s1), KRT5 (Hs00361185_m1), WT1 (Hs01103751_m1), PI3KCA (Hs00907957_m1), PIK3CD (Hs00192399_m1), AKT1 (Hs00178289_m1), AKT2 (Hs01086102_m1), AKT3 (Hs00987350_m1), BCL2 (Hs00608023_m1) and RPLP0 (Hs00420895_gH). The fold change in gene expression was calculated with the 2^−ΔΔCt method and the presented data were normalized to the mesothelium values.