Rna pcr kit

Total RNA was extracted using the TRIzol reagent (Invitrogen). First-strand complimentary DNA (cDNA) was synthesized from total RNA, according to manufacturer’s protocol from the RNA PCR kit (Promega, Madison, WI, USA). Quantitative PCR was carried out using the ABI 7300 real-time PCR system (Applied Biosystems, Carlsbad, CA). The primers are listed in the Additional file 1. The target mRNA expression was quantified and β-actin was used as an endogenous reference. For the list of primers, see Additional file 1: Table S2. Results were expressed as fold change in gene expression.

Cells were assigned to three groups and treated with three different chemoagents. After treatment, total RNA was extracted using the TRIzol reagent (Invitrogen). First-strand complimentary DNA (cDNA) was synthesized from total RNA according to the manufacturer’s protocol for the RNA PCR kit (Promega, Madison, WI, USA). Quantitative PCR was carried out using the ABI 7300 real-time PCR system (Applied Biosystems, Carlsbad, CA). EpCAM expression was quantified and β-actin was used as an endogenous reference. Results were expressed as fold change in gene expression.

Total RNA was extracted using Trizol reagent (RNA STAT 60 Tel-Test Ambion, Austin, TX, USA) and a Nanodrop ND-1000 spectrophotometer was used to quantify RNA concentration and purity. First-strand complimentary DNA (cDNA) was synthesized from total RNA using an RNA PCR kit (Promega, Madison, WI, USA), according to the manufacturer's protocol. Reverse transcription was performed in a Mastercycler Gradient (Eppendorf, Hamburg, Germany) using 1 µg of total RNA in a 20 μL volume containing 4 μL 25 mM MgCl₂, 4 μL AMV reverse transcriptase 5 × buffer, 2 μL dNTP, 0.5 μL RNase inhibitor, 1 μL of AMV reverse transcriptase, 1 μL of oligo (dT) primer, and nuclease-free water at 42°C for 50 min, followed by 95°C for 5 min. Primers for NQO1 (Mm01253561), HMOX1 (Mm00516005), and ACTB (Mm00607939) were purchased from Applied Biosystems (Carlsbad, CA, USA). qRT-PCR was performed in duplicate in 20 μL volumes containing 10 μL of TaqMan Universal PCR master mix, 9 μL of cDNA, and 1 μL of primer on an ABI 7500 Real-Time PCR system (Life Technologies Corp., Carlsbad, CA, USA). The comparative cycle time (Ct) method was used to determine the fold differences between the samples with respect to the amount of target present, which was normalized to ACTB as an endogenous reference, and relative to a calibrator (2^-△△Ct).