Topreal qpcr 2 premix sybr green with high rox

To confirm the detection limit of the template DNA of H. glycines in the qPCR assay, a sensitivity qPCR test with the primer set was performed. The template DNA (H. glycines) concentration was adjusted to 10 ng per μl by adding sterile distilled water to the DNA solution. The solution was diluted stepwise by 0.1-fold and DNA solution at five concentrations (10 ng, 1 ng, 100 pg, 10 pg, and 1 pg of DNA per microliter) was prepared. The PCR reaction mixture was composed of 10 μl of qPCR premix (TOPreal qPCR 2× Premix - SYBR Green with high ROX, Enzynomics), 7 μl of sterile distilled water (DNase, RNase free), 1 μl of template DNA (10 ng, 1 ng, 100 pg, 10 pg, and 1 pg per a reaction), 20 pmol of forward primer, and 20 pmol of reverse primer. The qPCR amplification and melting curve analysis were performed at the above qPCR assay conditions, and technical replicates were carried out in triplicates.

The WT and Actb-MBS MEF cells were cultured at a density of 2.2 × 10⁶ cells per 100 mm dish followed by the addition of 100 μM DRB (5,6-Dichlorobenzimidazole 1-β-D-ribofuranoside)(Sigma). Total RNA was extracted from cells at different time points of the DRB treatment using the TRIzol reagent (Invitrogen) and then treated with DNase I (Roche) at 37°C for 20 min to digest any genomic DNA followed by addition of 8 mM EDTA and heat-inactivation at 75°C for 10 min. cDNA was prepared from the total mRNA by reverse transcription using TOPscript Reverse Transcriptase enzyme (enzynomics) with 100 μM random hexamers. The reaction was done with the SimpliAmp Thermal Cycler (Applied Biosystems) using the following cycling conditions: annealing at 25°C for 10 min, reaction at 42°C for 60 min and inactivation at 95°C for 5 min.
A 2-step RT-qPCR reaction was done with 100 μM of each primer (mouse β-actin: FW 5′-CCACTGCCGCATCCTCTTCC-3′, REV 5′-CTCGTTGCCAATAGTGATGACCTG-3′; mouse GAPDH: FW 5′-CATGGCCTTCCGTGTTCCTA-3′, REV 5′-GCGGCACGTCAGATCCA-3′) and TOPreal qPCR 2× PreMIX (SYBR Green with high ROX) (enzynomics) using the StepOnePlus Real-Time PCR System (Thermo Fisher). Samples were run in triplicates and analyzed using the StepOnePlus Real-Time PCR software. β-actin mRNA levels were analyzed by the 2^−ΔΔCt method (Livak method) and normalized to the GAPDH mRNA levels.

To confirm the amplification of DNA extracted from single J2 of H. glycines (code: 100), a qPCR assay was conducted with the developed species-specific primer set. The qPCR reaction mixture was composed of 10 μl of qPCR premix (TOPreal qPCR 2× Premix - SYBR Green with high ROX, Enzynomics), 7 μl of triple distilled water (DNase, RNase free), 1 μl of template DNA, 20 pmol of forward primer, and 20 pmol of reverse primer. Six biological replicates were carried out and a positive control was designed for the reaction using DNA extracted from a single cyst of H. glycines (code: 100). The reactions were performed with the same qPCR machine and conditions as above. The qPCR products were confirmed using a UV transilluminator (UVCI-1100, Major Science).