Validating Microarray Findings via RT-PCR

For the present study, additional real-time RT-PCR was performed to validate additional genes from several major functional classes altered by injury. The same animal samples and RNA extractions used for microarray analyses were used for RT-PCR. RT-PCR was performed for the following genes: PTGES, PLA2GA2, PLA1A, GDF10, TNC, ASPN, TIMP1, FABP4, LCN2, IL1B, EMR1, PLTP, MOBP, COMT, CRYAB, ARSB, NAAA, PTPRC, AXL and PTAFR. cDNA synthesis was performed using 100 ng total RNA and the TaqMan Reverse Transcription Reagents kit (Applied Biosystems, Carlsbad, CA, USA). Real-time PCR was carried out in a 7900 HT thermocycler (Applied Biosystems) using 2× Gene Expression Master Mix and Assays on Demand (Applied Biosystems). For comparative analysis, the 2^-δδCt method was used [86 (link)].