Biodoc analyzer

The expression of MCT1-4 and GLUT1-4 was tested in OLN-93 cells by reverse transcriptase polymerase chain reaction (RT-PCR). Five of the 15 dishes per experimental setting were pooled for these analyses.
Total ribonucleic acid (RNA) was isolated from OLN-93 cell cultures using guanidinium isothiocyanate/phenol/chloroform (peqGOLD TriFast™, peqlab, Erlangen, Germany). For removing deoxyribonucleic acid (DNA) contamination, 5 μg of the total cell RNA was treated with Turbo DNA-free (Ambion, Austin, TX, USA) according to the manufacturer's instructions. RNA (4.5 μl; 2.25 μg input RNA) was reverse transcribed using the RevertAid™ H Minus First strand cDNA Synthesis Kit primed with Oligo(dT)₁₈ primers (Fermentas, St. Leon-Rot, Germany; primers listed in the Table 1). cDNA (2 μL) was then PCR-amplified with Taq-DNA-polymerase (peqlab, Erlangen, Germany).
One-tenth of each reaction product was electrophoresed on a 1% agarose gel. The PCR product bands were quantified by densitometric analysis using a Biometra BioDocAnalyzer. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene for data normalization (GAPDH showed no medication-induced changes in expression), the ratio of MCT and GLUT expression to GAPDH expression was calculated.

Total RNA was extracted using TRIzol reagent according to the manufacturer's instructions, followed by DNase treatment (Thermo Fisher). RNA (2 µg) was used for first‐strand complementary DNA (cDNA) synthesis. Reverse‐transcription polymerase chain reaction (RT‐PCR) was performed with 200 ng cDNA, Taq DNA polymerase, and the primers listed in supplemental online Table 1. mRNA signals were quantified by densitometric analysis using a Biometra BioDoc‐Analyzer, and the ratio of their expression to that of a housekeeping gene (GAPDH) was calculated.