RNA was prepared by using Tri Reagent (Sigma-Aldrich, St Louis, MO, USA) and was reverse transcribed into cDNA using a Super Script II Reverse Transcriptase Kit (Invitrogen Inc., Carlsbad, CA, USA). PCR was performed on an ABI StepOne Plus System (Applied Biosystems, Foster City, CA, USA) using the LightCycler® First Start DNA Master SYBR Green I reagent (Roche Diagnostics, Basel, Switzerland). The mRNA level was normalized using the TATA-box binding protein (TBP) mRNA level as the standard. The following primers were used: GNMT forward, 5′-ACTGGATGACTCTGGACAA-3′ and reverse, 5′-ACTGAGGATGTGGTCGT-3′; TBP forward, 5′-TGCACAGGAGCCAAGAGTGAA-3′ and reverse, 5′-CACATCACAGCTCCCCACCA-3′.
Lightcycler first start dna master sybr green 1 reagent
Manufactured by Roche
Sourced in United States
The LightCycler® First Start DNA Master SYBR Green I reagent is a real-time PCR (polymerase chain reaction) reagent. It contains SYBR Green I, a fluorescent dye that binds to double-stranded DNA, enabling the detection and quantification of DNA amplification during the PCR process.
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3 protocols using lightcycler first start dna master sybr green 1 reagent
1
GNMT Expression Quantification by qRT-PCR
2
RNA Isolation and Gene Expression Analysis
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Total RNA was isolated by using Tri Reagent (Sigma-Aldrich) and cDNA was synthesized using a Super Script II Reverse Transcriptase Kit (Invitrogen Inc., Carlsbad, CA, USA). PCR was performed on an ABI StepOne Plus System (Applied Biosystems, Foster City, CA) with the LightCycler® First Start DNA Master SYBR Green I reagent (Roche Diagnostics, Basel, Switzerland). The mRNA level was normalized using the TBP as an internal control to calculate relative expression. The primers used in this study shown in Supplementary Table S2 .
3
Quantifying CYP1A Transcripts in Stable Cells
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RNA was prepared by using Tri Reagent (Sigma–Aldrich, St Louis, MO, USA) and was reverse transcribed into cDNA using a Super Script II Reverse Transcriptase Kit (Invitrogen Inc., Carlsbad, CA, USA). PCR was performed on an ABI StepOne Plus System (Applied Biosystems, Foster City, CA, USA) using the LightCycler® First Start DNA Master SYBR Green I reagent (Roche Diagnostics, Basel, Switzerland). The mRNA level was normalized using the GAPD mRNA level as the standard. The expression profiles of Cyp1a1 and Cyp1a2 mRNA in BaP treated GFP, GNMT or GNMTS9A stable cells were normalized to their DMSO control. The following primers were used: CYP1A1-F (5′-GCTGCAACGGGTGGAATT) and CYP1A1-R (5′-CAGGCATGCTTCATGGTTAGC-3′) for CYP1A1; CYP1A2-F (5′-GGAGCAGGATTTGACACAGTCA-3′) and CYP1A2-R (5′-TTCCTCTGTATCTCAGGCTTGGT-3′) for CYP1A2; GAPD-F (5′-TGGTATCGTGGAAGGACTCA-3′) and GAPD-R (5′-AGTGGGTGTCGCTGTTGAAG-3′) for GAPD.
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