Sybr green pcr mix

SYBR Green qPCR was performed in a final volume of 22.5 μL, with 12.5 μL of SYBR Green PCR MIX (Promega, Madison, USA), 0.5 μL of each primer (10 μM), 6.5 μL of ultrapure water and 2.5 μL of cDNA (1:4) in the Rotor Gene-Q® thermocycler (Qiagen, Hilden, Germany). Primer sequences that amplify fragments of the Dsg1, Dsg2 and Dsg3 genes and of the human ribosomal gene (18S) as endogenous genes were used.13 (link), 14 (link), 15 (link) The primer sequences, the resulting amplified product (amplicon) and the amplification cycle parameters are detailed in Supplemental Table 1. The relative gene expressions of Dsg1, Dsg2 and Dsg3 were calculated using the equation 2 ^(−ΔcT), having the 18S rRNA as the reference gene; ΔcT was calculated by subtracting the cycle threshold (cT) value from the target gene (Dsg1, Dsg2 or Dsg3) from the cT value of the reference gene (18S).¹⁶ (link) To verify the specificity of each sequence, the obtained amplicon was purified (ExoSAP-IT®), sequenced (3500 Genetic Analyzer, Applied Biosystems, USA), and analyzed at the NCBI BLAST.

cDNA quantification was performed by real-time PCR (DNA Engine Opticon 2; MJ Research, Ramsey, MN). Reactions were performed using a SYBR Green PCR mix (Promega, Fitchburg, WI, USA) and amplified according to the following thermal profile: initial denaturation (95 °C, 15 minutes) followed by 40 cycles of 15 seconds at 95 °C (denaturation) and 1 minute at 60 °C (annealing) and 20 seconds at 72 °C (extension). A Ct value of 40 or higher means no amplification and this value was not included in the calculations. Results were expressed as ΔΔCt and presented as ratio between the target gene and the GAPDH housekeeping mRNA. All the samples were analyzed in triplicate.

First-strand complementary DNAs (cDNAs) were synthesized from 1 μg of each RNA sample by Reverse Transcription System and quantified by real-time PCR with SYBR Green PCR Mix (Promega) on an Applied Biosystems 7300 RT-PCR cycler. The relative gene expressions were calculated by the delta-delta Ct method with β-Actin used as the reference gene. Absolute quantification of viral load was obtained by extrapolating the Ct value against a copy number standard curve. The primers are shown in the supplementary table.