PCR and qRT-PCR were performed as previously described (12 (link)). Briefly, a PCR kit (Qiagen, Valencia, CA) was used to genotype the SALL4B Tg founder mice and transmission of the transgene. Genomic DNA was purified from a mouse tail using a high-quality DNA kit (Gentra Systems, Minneapolis, MN). The human SALL4B primer sequences were showed in supplementary table 1 . Total RNA was isolated using a phenol-free and filter-based RNA isolation system (Qiagen) digested with DNase I to remove DNA contamination. Primer sequences for qRT-PCR were designed using Primer Express® software (Applied Biosystems, Foster City, CA) and were listed in supplementary table 1 . All reactions were performed in an ABI-7000 sequence detection system using TaqMan PCR core reagents according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). PCR amplification was carried out in a 50 μl final volume containing 1×PCR buffer, 1 μM of each primer, and 10 μl of RNA at 95°C for 10 min followed by 45 cycles at 95°C for 15 sec and then at 60°C for 1 min. For each sample, expression of the GAPDH gene was used to normalize the amount of investigated transcript.
Taqman pcr core reagents
Manufactured by Thermo Fisher Scientific
Sourced in United States
TaqMan PCR core reagents are a set of essential components for conducting Polymerase Chain Reaction (PCR) experiments. These reagents include the necessary enzymes, primers, probes, and buffers required to perform quantitative real-time PCR (qPCR) analysis.
Automatically generated - may contain errors
3 protocols using taqman pcr core reagents
1
Transgenic SALL4B Mouse Generation
2
Transgenic SALL4B Mouse Generation
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PCR and qRT-PCR were performed as previously described (12 (link)). Briefly, a PCR kit (Qiagen, Valencia, CA) was used to genotype the SALL4B Tg founder mice and transmission of the transgene. Genomic DNA was purified from a mouse tail using a high-quality DNA kit (Gentra Systems, Minneapolis, MN). The human SALL4B primer sequences were showed in supplementary table 1 . Total RNA was isolated using a phenol-free and filter-based RNA isolation system (Qiagen) digested with DNase I to remove DNA contamination. Primer sequences for qRT-PCR were designed using Primer Express® software (Applied Biosystems, Foster City, CA) and were listed in supplementary table 1 . All reactions were performed in an ABI-7000 sequence detection system using TaqMan PCR core reagents according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). PCR amplification was carried out in a 50 μl final volume containing 1×PCR buffer, 1 μM of each primer, and 10 μl of RNA at 95°C for 10 min followed by 45 cycles at 95°C for 15 sec and then at 60°C for 1 min. For each sample, expression of the GAPDH gene was used to normalize the amount of investigated transcript.
3
Absolute Quantification of Viral Genomes
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For absolute quantification of the viral genome, a standard curve was obtained using serial 10-fold dilutions (three copies to 3 × 107 copies in 10 μl) of purified genomic DNA of BAd-ΔE1E3. The copy number of the viral genome was calculated based on spectrophotometric quantification and the molecular mass of BAd-ΔE1E3’s genomic DNA. A standard curve was run for each set of assays. For qPCR, 10 μL of isolated DNA was used in a 25 μL reaction using Taqman PCR core reagents (Applied Biosystems, Foster City, CA, USA). The reaction mixture contained 10x Taqman buffer, 250 nM of forward and reverse primers, and 100 nM of Taqman probe along with other standard kit components. Each reaction was carried out in duplicate. The qPCR was performed using the Mx3000 Thermocycler (Stratagene, Cedar Creek, TX, USA) with the following reaction conditions: 50°C for 2 min, followed by polymerase activation (95°C for 10 min), 45 cycles of denaturation (95°C for 15 s) and annealing/extension (60°C for 1 min). The threshold cycle (Ct) value for individual reactions was determined, and data was analyzed with MxPro software to obtain the absolute copy number of viral genome.
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