Gotaq qpcr master mix system

Total RNA was extracted and genomic DNA was removed using the RNeasy Plus Mini Kit (Qiagen, Valencia, CA). Microarray analyses were performed by the Boston University Microarray and Sequencing Resource using GeneChip® Mouse Gene 2.0ST arrays (Affymetrix, Santa Clara, CA). For RT-qPCR analyses, cDNA was prepared from total RNA using the GoScript™ Reverse Transcription System (Promega), with a 1:1 mixture of random and Oligo (dT)15 primers. All qPCR reactions were performed using the GoTaq® qPCR Master Mix System (Promega). The qPCR reactions were performed using a 7500 Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA): hot-start activation at 95°C for 2 min, 40 cycles of denaturation (95°C for 15 sec) and annealing/extension (55°C for 60 sec). The primer sequences are provided in Table S1. Relative gene expression was determined using the Pfaffl method to account for differential primer efficiencies (Pfaffl 2001 (link)), using the geometric mean of the Cq values for beta-2-microglobulin (B2m) and 18s ribosomal RNA (Rn18s) for normalization. The Cq value from naïve, undifferentiated cultures was used as the reference point. Data are reported as “Relative Expression”, unless log-transformed and reported as “Log Relative Expression.”

Total RNA was extracted and genomic DNA was removed using the RNeasy Plus Mini Kit (Qiagen, Valencia, CA). cDNA was prepared from total RNA using the GoScript™ Reverse Transcription System (Promega), with a 1:1 mixture of random and Oligo (dT)₁₅ primers. All qPCR reactions were performed using the GoTaq® qPCR Master Mix System (Promega). Validated primers were purchased from Qiagen Inc. (Valencia, CA)(Table S1). Sequences for the Abca1 and Srebp1c primers, which were synthesized, were previously reported.^{35 (link)} qPCR reactions were performed using a 7500 Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA): Hot-Start activation at 95°C for 2 min, 40 cycles of denaturation (95°C for 15 sec) and annealing/extension (55°C for 60 sec). Relative gene expression was determined using the Pfaffl method,^{36 (link)} using the threshold value for 18s ribosomal RNA (Rn18s) for normalization. No significant differences were observed in the expression of Rn18s across the different times or treatments (data not shown). The Cq value from naïve, undifferentiated cultures was used as the reference point, and data were reported as “Fold Change from Naive.” Expression data from medium wells and Vh wells were averaged for each experiment and reported as “Med/Vh.”

Following Nile Red staining, the staining solution was removed, and the cultures were washed two times with PBS. Then, total RNA was extracted and genomic DNA was removed using the RNeasy Plus Mini Kit (Qiagen, Valencia, CA). For RT-qPCR analyses, cDNA was prepared from total RNA using the GoScript™ Reverse Transcription System (Promega), with a 1:1 mixture of random and Oligo (dT)15 primers. All qPCR reactions were performed using the GoTaq® qPCR Master Mix System (Promega). Validated primers (18s ribosomal RNA (Rn18s): QT01036875, PPARγ1/2 (Pparg): QT00100296, fatty acid binding protein 4 (Fabp4): QT00091532, perilipin (Plin1): QT00150360, runt related transcription factor 2 (Runx2): QT00102193, Osterix (Osx): QT00293181, osteocalcin (Bglap): QT00259406O) were purchased from Qiagen. qPCR reactions were performed using a 7500 Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA). Relative gene expression was determined using the Pfaffl method (Pfaffl, 2001 (link)), using the threshold value for Rn18s for normalization. No significant differences were observed in the expression of Rn18s across the different treatments (data not shown). The Cq value from naïve, undifferentiated cultures was used as the reference point. Data were normalized to the expression in cultures treated with 100 nM Rosi and reported as “% Rosi Control.”