Mgb nfq quencher

DNA was extracted from tissue samples using QIAmp DNA FFPE Tissue kit according to manufacturer’s recommendations (Qiagen). 100 ng genomic DNA of each sample was used for quantitative PCR analysis performed in triplicates as previously described [22 (link)]. Primers amplifying MCV LT antigen (1052–1131 nt; forward: 5’-ctctgggtatgggtccttctca-3’, reverse: 5’-catggtgttcgggaggtatatcg-3’) and internal probe (5’-ccaggcttcagactcc-3’) labeled with FAM and MGB-NFQ quencher (Applied Biosystems) were used. Copy numbers were calculated by generating standard curves of Ct values obtained from serial dilutions of known concentrations of MCV DNA amplified by PCR. RNaseP (Applied Biosystems) was used to determine cell number. qPCR reactions were performed on the Bio-rad CFX96 Real-Time PCR system (Bio-rad) with UNG (+) TaqMan Universal PCR Master Mix II (Applied Biosystems). Amplification reactions of all target genes were performed with the following condition: 50°C for 2 min, denaturing at 95°C for 10 min, 40 cycles of 95°C for 15 sec and 60°C for 1 min. Results were expressed as numbers of viral copies per cell calculated from Ct values of viral and cellular gene standards.

DNA loss and recoveries from each experiment were ascertained via quantitative polymerase chain reaction (qPCR). Samples were amplified using SureStart Taq polymerase (Agilent, Santa Clara, CA, USA) using an Applied Biosystems 7500 thermocycler. Custom primers and Taqman probe sequences were designed for specific sequences of λ-DNA. The forward primer was (5'- GTG GAA TGA ACA ATG GAA GTC AAC AA -3'), the reverse primer was (5'- GGC AGA GTC ATA AAG CAC CTC ATT A -3') (Integrated DNA technologies), and the Taqman probe was (5'- AGG TGC TAC GGC GGC AGA GT -3') tagged with 6-FAM at the 5’-end and a MGB-NFQ quencher at the 3’-end (Applied Biosystems, Waltham, MA, USA). The resulting amplicon was 177 base pairs long. Each 25 μL reaction contained 5 μL of the purified DNA filtrates. Samples were placed in a 96-well plate (Applied Biosystems, Waltham, MA, USA), initially heated to 95°C to activate the polymerase and then cycled 40 times through 30 sec of 95°C for DNA denaturing, 5 sec of 65°C for primer annealing, and 30 sec of 72°C primer extension. qPCR generates curves of the relative fluorescence (ΔRn) after each temperature cycle. The amount of starting DNA is calculated based on the cycle at which the fluorescence in a given well reached a threshold value as shown in Fig 2.