Verso rt pcr kit

Total cellular RNA was harvested using TRIZOL Reagent (Invitrogen) and after DNase I digestion (Promega), cDNA synthesized in a 20-μl reaction volume from 500 ng RNA using Verso RT-PCR Kit containing RT Enhancer to remove contaminating DNA (ABgene). Quantitative real-time PCR was performed in triplicate using 5 μl diluted cDNA, 1 μl primers/probe mix with Taqman detection chemistry and 10 μl 2 × ABsolute Blue QPCR Mix (ABgene). Reactions were performed for 40 cycles (95 °C for 15 s, 60 °C for 30 s, and 72 °C for 15 s) using a Rotor-Gene 6000 instrument (Corbett Life Science). A standard curve was generated in each run to quantify the amount of amplicons. Amounts of unspliced precursor RNA and spliced mRNA were normalized to EGFP mRNA for transfection efficiency and to β-actin mRNA as internal normalizing standard [10 (link)]. Amplification efficiency of spliced and unspliced forms, determined by CT slope, was similar and at least 99%.

Vectors expressing cell-surface CD28, CD28 fused C-terminally to GFP, cell-surface B7-2 and B7-2 or B7-2C fused C-terminally to Cherry have been described (4 (link), 5 (link)). Vector expressing B7-1 was generated by cDNA synthesis of human CD80 (NM_005191.3) from total human PBMC RNA using Verso RT-PCR kit (ABgene). CD80 cDNA was generated using KOD polymerase (Novagen) with phosphorylated PCR primers 5′-GAC GTC GAC ATG GGC CAC ACA CGG AGG and 5′-CAC GCG GCC GCT TAT ACA GGG CGT ACA CTT TC CC. The PCR product was inserted into pEGFP-N3 DNA (Clontech) that had been digested with SalII and NotI and lacked the GFP region, using Fast-Link DNA Ligation Kit (Epicenter). Vector expressing B7-1 fused C-terminally to Cherry was generated from B7-1 cDNA vector template with phosphorylated PCR primers 5′-TAC TCG AGA TGG GCC ACA CAC GGA GG and 5′-GTC CGC GGT ACA GGG CGT ACA CTT TCC CT TC, deleting the B7-1 termination codon. Upon digestion with XhoI and SacII, the PCR product was inserted into pmCherry-N1 DNA (Clontech).

DNA-free total RNA was extracted from LV samples by a standard protocol with an EZ-RNA-total RNA Isolation Kit (Biological Industries, Israel). cDNA synthesis (500 ng) was carried out with the Verso RT-PCR Kit (ABgene, USA) according to the manufacturer’s instructions. qRT-PCR was performed on a Bio-Rad iQ-Cycler using a SYBR Green PCR kit (Invitrogen, Israel). The primer sequences are provided in S1 Data. Western blot was performed according to standard procedure [3 (link)] using the following antibodies: mouse monoclonal anti-Cytochrome C (Cyt C) (Cat # ab14734, Abcam, USA), mouse monoclonal anti-Voltage-dependent anion channel (VDAC)1/porin (Cat # ab110325, Abcam, USA), and mouse monoclonal anti- sirtulin (Sirt)3 (Cat # Sc-365175, Santa Cruz Biotechnology, USA), rabbit anti-GAPDH (Cat # 37168, Abcam, USA) followed by goat anti-mouse horseradish peroxidase (Cat # 10015289, Jackson ImmunoResearch Laboratories, Inc. USA) and goat anti-rabbit horseradish peroxidase (Cat # 111035003, Jackson ImmunoResearch Laboratories, Inc. USA)