Steponeplus real time pcr system

Semi-quantitative RT-PCR for targets of capsid, nsP3, and 18S used thermal cycling conditions adapted from Verso 1-step RT-qPCR kit (AB4101C, ThermoFisher) per the manufacturer’s instructions: 1 cycle at 50 °C for 20 min, 1 cycle at 95 °C for 15 min, 40 cycles at 95 °C for 15 s with 51 °C (capsid), 59 °C (nsP3), 60 °C (18S) for 1 min using StepOnePlus™ Real Time PCR system. qRT-PCR for detection of viral negative-strand used thermal cycling conditions adapted from PowerUp SYBR Green (A25742, ThermoScientific) per the manufacturer’s instructions: 1 cycle at 50 °C for 2 min, 1 cycle at 95 °C for 2 min, 40 cycles at 95 °C for 15 s, 60 °C for 15 s and 72 °C for 1 min using StepOnePlus™ Real Time PCR system. A no template cDNA control and mock infections were included for all analyses and established the limits of detection. Semi-quantitative RT-PCR values were calculated using the ΔΔCt method [39 (link)] with viral entities normalized to 18S levels.

Total RNA was isolated from indicated tissues and cells by Trizol reagent (Invitrogen, Thermo Fisher Scientific) in accordance with the manufacturer’s manual. The first strand cDNA was generated using the RNA and the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara, Dalian, China). Real-time PCR was performed using TB Green^® Premix Ex Taq™ II (Takara) on StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) with the primers 5’-GGCTCCTTGCTACTTTTAGGG-3’ (forward) and 5’-TGGGGGGGGTCTTTCTCTTC-3’ (reverse) for FUT8-AS1, 5’-GAAATACGCCAGTACCGAATG-3’ (forward) and 5’-TTCTCCTCCAGGGAAGTCAG-3’ (reverse) for NRAS, 5’-GTCGGAGTCAACGGATTTG-3’ (forward) and 5’-TGGGTGGAATCATATTGGAA-3’ (reverse) for GAPDH. GAPDH was used as endogenous control for the quantification of FUT8-AS1 and NRAS expression. For the quantification of miRNAs and pri-miRNAs expression, real-time PCR was carried out using the TaqMan™ Advanced miRNA Assay (Thermo Fisher Scientific) and TaqMan™ Pri-miRNA Assay (Thermo Fisher Scientific) respectively on StepOnePlus Real-Time PCR System in accordance with the manufacturer’s manuals.

Total RNA extraction was performed using TRIzol® Reagent (Thermo Fisher Scientific) and purified using a RNeasy MinElute^® Cleanup Kit (QIAGEN, Germany) as previously reported^{21 (link)}. Purified total RNAs were quantified using an ND-1000 spectrophotometer (Thermo Fisher Scientific). Reverse transcription was carried out using a StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific). The expression levels of miRNAs were quantified using a TaqMan MicroRNA assay kit (Thermo Fisher Scientific). Twenty-five femtomoles of synthetic Caenorhabditis elegans miRNA (cel-miR-39, Thermo Fisher Scientific, USA) were added to each sample as the internal control. The miRNA expression was quantified using the TaqMan MicroRNA assays kit (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol. QRT-PCR reactions were performed on the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, USA) using a standard protocol (95°C 10min 1cycle; 95°C 15s, 60°C, 1min, 40 cycles). Each sample were run in triplicate. The fold expression of the target gene relative to the averaged internal control gene in each sample will be calculated using the comparative threshold cycle (Ct) method and evaluated by 2^−△△Ct, △△Ct = Patient (Ct_{miRNAs gene} − Ct_cel-miR-39) − Mean of controls (Ct_{miRNAs gene} − Ct_cel-miR-39).

For miR RT-qPCR, total RNA was reverse transcribed with the TaqMan miR RT kit (Applied Biosystems, Life Technologies, Inc.) as described in the manufacturer’s protocol, with miR-specific, stem-loop primers (Applied Biosystems, Life Technologies, Inc.). TaqMan miR assays (Applied Biosystems, Life Technologies, Inc.) were used to detect mature miRs by the comparative C_T method with the StepOnePlus real-time PCR system (Thermo Fisher Scientific). Assay IDs 000390, 000391, 002367, and 000507 were used to detect miR-15b-5p, -16-5p, -193b-3p, and -203a-3p, respectively. RT-qPCR assays were performed in triplicate, and the noncoding small nuclear RNA (snRNA) U6 (assay ID, 001973) was used as an endogenous small-RNA control.
For RT-qPCR of miR targets, following RNA isolation, total RNA was DNase treated with the TURBO DNA-free kit. DNase-treated total RNA was then reverse transcribed with TaqMan RT reagents (Life Technologies, Inc., Applied Biosystems). TaqMan assays for TP63 (assay ID, Hs00978343_m1) and BMI1 (assay ID, Hs00995536_m1) were used to detect targets by the comparative C_T method with the StepOnePlus real-time PCR system (Thermo Fisher Scientific). RT-qPCR assays were performed in triplicate, and 18S rRNA was used as an internal control.

Bisulfite-treated DNA served as template for amplification of methylated LINE-1 promotor sequences in a normalized real-time MS-PCR for genetically imbalanced DNA specimens as described^{15 (link),17 (link),22 ,44 (link)}. Quantification was done using StepOne Plus Real Time PCR System (Applied Biosystems, Foster City, United States of America) with the following primers for sL1met: 5′-GCGCGAGTCGAAGTAGGGC-3′; asL1met:5′-CTCCGACCAAATATAAAATATAATCTCG-3′. Amplification of an adjacent region without CpGs was used to normalize the amount of LINE-1 methylation. The primers used for reference amplification were for sLcontrol: 5′-AGGTTTTATTTTTGGGGGTAGGGTATA-3′; asL1control:5′-CCCCTACTAAAAAATACCTCCCAATTAAAC-3′^{15 (link)}.
The following annealing temperatures were chosen: L1met: 61 °C; L1control: 58 °C. The amplification conditions were denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, TM for 40 s, and 72 °C for 15 s. All reactions were run in triplicates on a Step One Plus Real-Time PCR System (Thermo Fisher Scientific, Massachusetts, USA). Relative differences in DNA methylation were calculated by the ΔΔCt-method.