Quantstudio real time pcr analyzer

Gene expression validation experiments were performed on RNA isolated from an independent set of the same 3D organoid lines exposed/unexposed to ethanol. RNA for quantative PCR (qPCR) was isolated essentially as described in RNA-Sequencing and Data Processing. RNA concentration was determined on a Qubit fluorometer (Thermo-Fisher). One microgram of Total RNA was Reverse Transcribed to first-strand cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo-Fisher). First-Strand cDNA was used for Taq-Man qPCR monitored on a QuantStudio Real-Time PCR analyzer (Thermo Fisher). Pre-Designed TaqMan Gene Expression Assays (Thermo Fisher) were used for quantification of ANPEP, ARHGAP32, KIF16B, GNL3, ECE1 and the control gene HPRT1: Hs00174265_m1, Hs00206951_m1, Hs00402541_m1, Hs00205071_m1, Hs01043735_m1 and Hs02800695_m1 respectively.

Gene expression validation experiments were performed using RNA isolated from six matched colon organoid lines grown in CV and complex media. Lines were chosen based upon remaining quantities of RNA following RNA sequencing. RNA for qPCR was isolated as described above. RNA concentration was determined with a Qubit fluorometer (Thermo-Fisher, Waltham, MA, USA). A minimum of 500 ng of total RNA was reverse-transcribed to first-strand cDNA using a High-Capacity cDNA Reverse Transcription Kit (Thermo-Fisher, 4368814). First-Strand cDNA was used for Taq-Man qPCR monitored with a QuantStudio Real-Time PCR analyzer (Thermo Fisher). Pre-Designed TaqMan Gene Expression Assays (Thermo Fisher) were used for quantification of several genes. Beta glucuronidase (GUSB) was used as a control gene to determine delta-CT values. These values were then used as input for a linear regression analysis while accounting for sample pairing. Known markers of stem cells (LGR5 and MEX3A) and proliferation (MKI67) were tested for differences in expression between CV and complex media.