Total RNA was extracted from J774A.1 cells for each group in triplicate using Qiagen's RNeasy Mini Kit and was reverse transcribed using iScript cDNA Synthesis Kit (BioRad Laboratories, Hercules, CA, USA). Polymerase chain reaction was performed using Piko-Real (Thermofisher, Fremont CA, USA) for RNA expression levels of genes of interest, namely, lactate dehydrogenase A and B isoforms (LDHA/B) and monocarboxylate transporters 1 and 4 isoforms (MCT1/4), using Taqman probes (Applied Biosystems, Foster City, CA, USA) as described previously 11 (link). β-actin was used as the housekeeping gene, and the relative difference from the control cells was calculated for each primer/probe combination.
Protocol detail
Quantification of Metabolic Gene Expression
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Actin
Cdna
Cells
Genes
Housekeeping gene
Isoforms
Lactate dehydrogenase a
Monocarboxylate transporters 4
Polymerase chain reaction
Primer
Rna expression
Synthesis
Corresponding Organization : University of California, San Francisco
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Variable analysis
independent variables
- Cell type (J774A.1 cells)
- RNA expression levels of lactate dehydrogenase A and B isoforms (LDHA/B)
- RNA expression levels of monocarboxylate transporters 1 and 4 isoforms (MCT1/4)
- RNA extraction using Qiagen's RNeasy Mini Kit
- Reverse transcription using iScript cDNA Synthesis Kit (BioRad Laboratories)
- Polymerase chain reaction using Piko-Real (Thermofisher)
- Use of Taqman probes (Applied Biosystems) for gene expression analysis
- β-actin as the housekeeping gene
- Positive control: Not specified
- Negative control: Not specified
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