Qpcrsoft 4

Cells were grown close to confluence in T25 flasks before being incubated (or not as a control) for 2 h with 9.25 MBq/mL of ^99mTc-BBN and ^99mTc-TPP-BBN. After incubation, the cells were detached, washed once with PBS and resuspended in 200 µL PBS. Then, total DNA was isolated using the “DNeasy Blood and Tissue Kit” (Qiagen), according to the manufacturer’s instructions. DNA concentration and purity were determined using a μDrop™ plate in a Varioskan™ LUX microplate reader (Thermo Fisher Scientific), and the samples were stored at −20 °C until further use. Only samples with high purity were used for the qPCR. qPCR was carried out in 96-well plates using the qTower3 system (Analytik Jena GmbH). Each well contained a reaction volume of 20 μL, which was composed of: 6.25 ng of total DNA, 1 × KAPA2G Fast HotStart ReadyMix (Roche), 1 × fluorescent dye EvaGreen (Biotium) and 500 nM of the forward and reverse primers (StabVida). PCR efficiencies were calculated using the qPCRsoft 4.1 (Analytik Jena GmbH) by preparing template dilutions corresponding to 12.5, 6.25, 3.125 and 1.5625 ng of total cellular DNA. The presence of the specific amplification products was confirmed through agarose gel electrophoresis. The results were used to calculate the number of copies of mitochondrial DNA as previously described [68 (link)], assuming a ploidy of 3.3 for the PC3 cell line [69 (link)].

Total RNA isolation and quantitative RT-PCR analysis were performed as described previously [23 (link)]. All samples were measured in triplicate and normalized to two housekeeping genes (Dpm1 and Txnl4a). The qPCR results were obtained and evaluated with the software qPCRsoft 4.1 (V4.1.3.0, Analytik Jena, Jena, Germany), and then analyzed using the standard delta delta Cq method.

Reverse transcription (RT) into complementary DNA (cDNA) was conducted using Maxima Reverse Transcriptase (EP0752, Thermo Fisher Scientific) and Oligo(dT)18 primer (SO131, Thermo Fisher Scientific). Quantitative PCR (qPCR) was performed using innuMIX qPCR SyGreen Sensitive (845‐AS‐1310200, Analytik Jena) on a qTOWER 3 G touch thermal cycler (Analytik Jena). Primers for RT‐qPCR were designed using NCBIs PrimerBLAST (https://www.ncbi.nlm.nih.gov/tools/primer‐blast/). If possible, primer pairs (Table S1) were designed to span an exon‐exon junction, thus being mRNA‐specific. If this was not possible they needed to be separated by at least one intron. Primers were validated using no template controls, no RT controls, agarose gel electrophoresis and melt curve analysis. Amplification data were analyzed using qPCRsoft 4.0.8.0 (Analytik Jena). Normalized expression values were calculated using the Pfaffl method considering amplification efficiency values determined by standard curves.

We performed reverse transcription with Maxima Reverse Transcriptase (EP0752, Thermo Fisher Scientific) and Oligo(dT)18 primers (SO131, Thermo Fisher Scientific) according to the manufacturer’s instructions. innuMIX qPCR SyGreen Sensitive (845-AS-1310200, Analytik Jena, Jena, Germany) was used on a qTOWER 3 G touch thermal cycler (Analytik Jena) for quantitative PCR (qPCR) of cDNA. qPCR primers were designed with NCBIs PrimerBLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). For mRNA specificity, qPCR primers were designed to span an exon-exon junction where possible. Primers were tested with no template controls, original RNA, and melt curve analysis. Primer sequences are listed in Additional file 2: Table S4. qPCR output files were analyzed in qPCRsoft 4.0.8.0 (Analytik Jena) and normalized expressions (ddCt algorithm) were calculated using the reference genes Gak and Srp72.

With the thermal shift assay, thermal denaturation of the investigated protein was accessed as previously described [48 (link)]. The fluorogenic dye Sypro orange was mixed with the protein and heated stepwise. When the protein is folded correctly, the dye does not bind efficiently on the hydrophilic surface. When hydrophobic stretches are exposed due to denaturation of the protein, binding of the dye leads to an increase in fluorescence. For the assay, 10 µl protein samples (> 0.1 mg/ml) are mixed with 5 µl 50x SYPRO Orange (Sigma Aldrich) and 20 µl 10 mM HEPES pH 8.0. As a positive control, 10 mg/ml lysozyme (Serva) was used. Measurement was done with qTower3G and qPCRsoft 4.0 (Analytik Jena) using the TAMRA Channel (λ_ex = 535 nm, λ_em = 580 nm). The heating program was 25 to 95 °C with steps of 2 °C, 120 s hold time per temperature and a heating speed of 4.4 °C/s.

Total RNA was isolated from cells using Trizol reagent (TIANGEN Biotech, Beijing). Five hundred nanograms of purified RNA was reverse transcribed using the Hifair 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (Y Cat. ID:KR116, TIANGEN Biotech). PCR samples were prepared with diluted cDNA (1:30), 5 μl SYBR Green PCR master mix (TIANGEN Biotech), .2 μM each of the forward and reverse primers (Table S2) in a total volume of 10 μl. Reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) was performed by using an Analytikjena qPCRsoft 4.0 (Germany). The relative expression level of the target gene was calculated by using the 2−ΔΔCT method and graphed as fold change (2−ΔΔCT) from control.

Sequences of the primers are presented in Table 3. Total RNA was extracted from CRC tissues by using TRIzol™ reagent [Yeasen Biotechnology (Shanghai) Co., Ltd., Shanghai, China] in accordance with the manufacturer's instructions. The cDNA was generated using the reverse transcription kit (Vazyme Biotech Co., Ltd., Nanjing, China). The qPCR test was performed using SYBR^® mix (Vazyme Biotech Co., Ltd.) using GAPDH for normalization. Analytikjena qPCR soft 4.0 (Germany) was used to run qPCR reaction program as follows: predenaturation temperature was 95 °C, 3 min; denaturation temperature was 95 °C, 10 s; annealing temperature was 60 °C, 59 s; a total of 40 cycles. The 2^−ΔΔCt method was used to calculate the relative expression levels of target genes.