Sybr green luna universal qpcr master mix

Plasmids pIRES2-EGFP and PX330 were kindly provided by Prof. Zhanjun Li (Jilin University, China). Plasmid pEGFP-MNDAL-N1 was constructed previously in our lab [2 (link)].
FITC-labeled goat anti-rabbit IgG (H + L), HRP-labeled goat anti-mouse IgG (H + L), HRP-labeled goat anti-rabbit IgG (H + L), DAPI, Enhanced BCA Protein Assay Kit, BeyoECL Plus kit, Cell lysis buffer for Western and IP, and Caspase3/8/9 ELISA Kit were purchased from Beyotime (Shanghai, China). Cell Counting Kit-8 (CCK-8) was purchased from Bioster (Wuhan, China).
TRIM21 rabbit polyclonal antibody, mouse P53 monoclonal antibody, and mouse Bcl-2 polyclonal antibodies were purchased from Proteintech (Wuhan, China). Lipofectamine 3000 Transfection Reagent and PTI-MEM were from Thermo Scientific (USA). Porcine TNF-α and IL-6 ELISA Kits were purchased from Absin (Shanghai, China). Rabbit PCV2 Cap antibody was from Biorbyt (Wuhan, China), and the rabbit anti-PCV2 Cap was prepared previously in the lab (1:200) [26 (link)]. Mouse IFN-α antibody, rabbit IFN-β antibody, mouse IFN-γ antibody, and mouse MNDA antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). TIANamp Virus DNA/RNA Kit, TRNzol-A⁺ Reagent, 2× Taq plus PCR Mix, and FastKing-RT SuperMix kit were from Tiangen (Beijing, China). Luna^® Universal qPCR Master Mix (SYBR Green) was from New England Biolabs (NEB, Ipswich, MA, USA).

Total RNA extraction was performed using Maxwell^® RSC (Promega, Fitchburg, MA, USA), and GoScript™ Reverse Transcriptase (Promega, Fitchburg, MA, USA) was used to obtain cDNA according to the manufacturer’s protocol. Sequences of qPCR primers used were PDK4 Fw: 5′-TCT GAG GCT GAT GAC TGG TG-3′; Rv: 5′-CAG GAA GCA GCA CTG GTG TA-3′ and GAPDH FW: 5′-GTC TCC TCT GAC TTC AAC AGC G-3′; Rv: 5′- ACC ACC CTG TTG CTG TAG CCA A-3′. qPCR was performed using an ABI StepOnePlus thermocycler (Applied Biosystems, Foster City, CA, USA) with Luna Universal qPCR Master Mix (SYBR Green) purchased from NEB (Ipswich, MA, USA).

RNA was isolated according to the RNA Cleanup Kit (NEB, T2030L) protocol, cDNA was prepared using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814), and qPCR was performed using the SYBR Green Luna Universal q-PCR Master Mix (NEB, M3003) on the QuantStudio 7 Flex Real-Time PCR System from Applied Biosystems (4485701). First, the efficiency of 18S primers and of each primer set for each gene of interest (GOI) were determined by computational standard curve analysis (QuantStudio 7 Flex Software). Relative efficiencies were calculated by dividing GOI by 18S RNA; as a base for dCT equations, the square of this value was used. For data analysis, we computed the square of the base minus dCT for each qPCR reaction. Primers were designed with the National Center for Biotechnology Information primer-designing tool and are listed in Supplemental Table 2.

In order to validate some of the specific targets identified by prediction analysis, we decided to concentrate further on processes of lung fibrogenesis and graft tolerance, such as STAT3, SMAD3 and SMAD4 [24 (link),25 (link),26 (link),27 (link)]. RNA was isolated from PBMCs derived from 4 BOS patients, prior to the start and after 6 months of ECP treatment, by miRNeasy Mini Kit (Qiagen). MiR-155-5p and miR-23b-3p expression levels were evaluated as described above. To evaluate STAT3, SMAD3 and SMAD4 gene expression, cDNA was retro-transcribed from 1 µg of total RNA using LunaScript RT SuperMix Kit (NEB). Relative levels of STAT3, SMAD3 and SMAD4 mRNA were assessed using SYBR^® Green Luna^® Universal qPCR Master Mix (NEB) and normalized to the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Each experimental condition was performed in triplicate. Relative gene expression level quantification was compared with internal standards and analyzed using the 2^−ΔΔCt method.

The obtained organs (bladder, muscle, liver, heart, lung, spleen, kidney, blood, brain, ear, tail, ovary, tooth, and bone marrow) were washed with 1 × PBS prior to genomic DNA extraction. Genomic DNA was extracted using an automated system (Maxwell® RSC, Promega, Madison, WI, USA) and a DNA extraction kit (Maxwell® RSC tissue DNA kit). These steps were performed according to the manufacturer’s instructions. Real-time PCR was performed using an ABI Step One Plus detection system (Applied Biosystems, Foster City, CA, USA) with SYBR Green (Luna Universal qPCR Master Mix, New England BioLabs Inc., Ipswich, MA, USA). The primer sequences used for the human Alu primer were as follows: forward 5′ CTG GGC GAC AGA ACG AGA TTC TAT 3′; reverse 5′ CTC ACT ACT TGG TGA CAG GTT CA 3′. qPCR was performed with 38 cycles of denaturation at 95 °C for 5 s and annealing at 60 °C for 20 s.

Total RNA was isolated from cells using miRNeasy Mini Kit (Qiagen). RNA concentration and purity were evaluated using a spectrophotometer (Nanodrop 2000, Thermo Scientific, Madison, WI, USA). cDNA was retrotranscribed from 1 µg of total RNA using LunaScript RT SuperMix Kit (NEB), according to the manufacturer’s instructions. Relative levels of Alpha Smooth Muscle Actin (ACTA2) and E-Cadherin (CDH1) mRNA were assessed using SYBR^® Green Luna^® Universal qPCR Master Mix (NEB) and normalized to the levels of glyceraldehyde-3- phosphate dehydrogenase (GAPDH) mRNA. All reactions were performed on an LC480 Real-Time PCR system (Roche Diagnostics, Vienna, Austria) according to the manufacturer’s recommendations. Each experiment was performed in triplicate. The threshold cycle (Ct) was defined as the fraction cycle number at which fluorescence exceeded the given threshold. Relative gene expression level quantification was compared with internal standards and analyzed using the 2^−∆∆Ct method.