Total RNA was obtained from R182 and TARA cell pellets using the Total RNA Kit from IBI Scientific (Dubuque, IA, USA). RNA yield and quality was assayed using absorbance spectra on a NanoDrop 2000 (Thermo Fisher). cDNA was reverse transcribed using the qScript cDNA synthesis kit from Quantabio (Beverly, MA, USA). Quantitative PCR was run on an Applied Biosystems Quantstudio 5 qPCR machine (Thermo Fisher), using PerfeCTa SYBR Green FastMix and Low ROX (Quantabio) in 10 μL reactions. PCR was run for 40 cycles, followed by melt curve analysis. Beta actin was used as a housekeeping gene. Relative gene expression levels were calculated using the 2−ΔΔCt method. Primers were obtained from Integrated DNA Technologies (Coralville, IA, USA), and sequences were as follows: BRCA2-Fd, 5′-AAAACGTTGAGCTGTTGCCA-3′; BRCA2-Rv, 5′-TGTGTTTTGGTTGAATTGTACCTT-3′; RAD51 Fd, 5′-CCAGACCCAGCTCCTTTACC-3′; RAD51 Rv, 5′-CACTGCGACACCAAACTCATC-3′; BRCA1 Fd, 5′-GGCTATCCTCTCAGAGTGACATTT-3′; BRCA1 Rv, 5′-GCTTTATCAGGTTATGTTGCATGGT-3′; Actin Fd, 5′-TTCCTGGGCATGGAGTCC-3′; and Actin Rv, 5′-CAGGTCTTTGCGGATGTCC-3′.
Perfecta sybr green fastmix and low rox
Manufactured by Quantabio
PerfeCTa SYBR Green FastMix and Low ROX is a pre-formulated, ready-to-use master mix for real-time PCR and quantitative PCR (qPCR) applications. It contains SYBR Green I dye, hot-start Taq DNA polymerase, and low ROX passive reference dye.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Quantstudio 5 qpcr machine, by Thermo Fisher Scientific (1 mentions)
Qscript cdna synthesis kit, by Quantabio (1 mentions)
Iscript reverse transcription supermix for qrt pcr, by Bio-Rad (1 mentions)
Quantstudio 6 flex system, by Thermo Fisher Scientific (1 mentions)
2 protocols using perfecta sybr green fastmix and low rox
1
Quantitative Analysis of DNA Repair Genes
2
Quantitative RT-PCR Analysis of Omomyc-RFP Expression
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Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed as previously described (Massó-Vallés et al. 2022 (link)). Briefly, to mimic the microarrays conditions, A375 and SkMel147 Omomyc-RFP cells were treated with 1 μg/mL doxycycline for 6, 12, and 24 h and for 4 d or left untreated. RNA was then extracted with TRIzol and quantified using NanoDrop. Equal amounts of RNA were reverse-transcribed to generate cDNA using iScript reverse transcription Supermix for qRT-PCR (Bio-Rad). SYBR Green qRT-PCR analysis was then performed on these cDNA samples with PerfeCTa SYBR Green FastMix and Low Rox (Quantabio) using the QuantStudio 6 FLEX system (Applied Biosystems). The data thus obtained were analyzed following the comparative (ΔΔCt) method described by Livak and Schmittgen (2001) (link). Analysis of relative gene expression data was performed using real-time quantitative PCR and the 2−ΔΔC(T) method (Livak and Schmittgen 2001 (link)). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-Tubulin were used as housekeeping genes. Sequences of primers used are listed in Supplemental Table S12 .
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