Transcription first strand cdna synthesis kit

RNA was isolated from cells using the High Pure RNA isolation Kit (Roche). The cDNA was synthesized with the Transcription First Strand cDNA Synthesis Kit (Roche). For semiquantitative PCRs 100 ng cDNA was used and GAPDH was co-amplified as a control. Real-time PCRs was performed using predesigned primers (Qiagen) and monitored using SYBR green fluorescence on a StepOnePlus Real-Time PCR system (Life Technologies). Target gene expression was calculated using the delta Ct method using GAPDH as internal control.

Total RNA was isolated from biopsy samples and cells using a RNeasy Mini Kit (QIAGEN, Hilden, Germany), and DNA digestion was performed using the Turbo DNA-free Kit (Thermo Fischer Scientific, Ireland), following the manufacturers’ instructions. For RNA extraction from the cell line experiments, the triplicates of each condition were extracted and pooled for gene expression analysis. cDNA was synthesized using 1 μg total RNA using Reverse Transcriptase enzyme provided by Transcription First Strand cDNA Synthesis Kit (Roche, Ireland). PCR primers (Eurofins, Ireland) and probes (Roche) were designed using the Universal ProbeLibrary Assay Design Center (https://www.roche-applied-science.com/sis/rtpcr/upl/adc.jsp; Roche Applied Science, Indianapolis, IN, USA). PCR reactions were performed in duplicates/triplicates by using the LightCycler 480 system (Roche Applied Science). Positive and negative controls were also included. The genes analyzed included CEACAM1, CEACAM3, CEACAM5, CEACAM6, CEACAM7, CEACAM20, CCL20, and IL-8/CXCL8 (see Supplementary Table 3 for primer and probe sequences). β-actin was used as housekeeping gene. The 2^−ΔΔ Ct method was used to calculate the relative changes in each of the genes relative to the expression of β-actin determined from real-time qRT-PCR experiments (28 (link), 29 (link)).

RNA extraction was performed using the NucleoSpin RNA extraction kit (Macherey-Nagel, Düren, Germany) according to manufacturer’s instructions. cDNA was generated by reverse transcription using the Transcription First Strand cDNA Synthesis Kit (Roche). qPCR reactions were set up with 400 ng of cDNA, 300 ng of each primer (HSPB1 5′-CTGACGGTCAAGACCAAGGATG-3′ (forward) and 5′-GTGTATTTCCGCGTGAAGCACC-3′ (reverse); HSP90AA1 5′-GTCCTGTGCGGTCACTTAGC-3′ (forward), and 5′-AAAGGCGAACGTCTCAACC-3′ (reverse)), and 1X Fast Start SYBR Green Master Mix (Roche). qPCR was performed using the 7300 Real Time PCR System and corresponding manufacturer’s software (Applied Biosystems, Carlsbad, CA, USA). Relative gene expression was normalized to 18S rRNA. Primers were synthesized at Eurogentec (Seraing, Belgium).

RNA was extracted automatically from 200-µL serum samples with the Magna Pure LC 2.0 System (Roche Diagnostics, Germany) following the total NA protocol. Complementary DNA (cDNA) was obtained by reverse-transcription with random hexamers using a Transcription First Strand cDNA Synthesis kit (Roche Diagnostics, Mannheim, Germany), following the manufacturer’s recommendations.

The mycelium samples collected during bioreactor cultivations were filtered and washed with cold water prior to freezing in liquid nitrogen. Samples were stored at −80 °C before extraction of RNA. Total RNA was extracted using the RNeasy Mini Kit (Qiagen). DNAse treatment (DNase I RNase-free, Thermo Fisher Scientific) was performed to remove residual genomic DNA from the RNA samples. Transcription First Strand cDNA Synthesis Kit (Roche) was used for the cDNA synthesis according to manufacturer’s instructions.