Qpcr human reference cdna

The relative gene expression levels for L1CAM were determined by the standard curve method, as previously described.^{14 (link)} Standard curves and linear equations were generated using 5-fold serial dilutions of random-primed qPCR Human Reference cDNA (Takara Bio, Clontec), which was prepared from a mixture of total RNAs collected from normal adult tissues. Within the range analysed, all standard curves were linear with an acceptable correlation coefficient (R²). The extent of target gene expression was calculated from the standard curve, and the cDNA in each sample was quantitatively normalised with respect to the GAPDH gene, which served as an internal control. Finally, the target gene mRNA levels were expressed as respective gene ratios relative to GAPDH mRNA levels. Real-time PCR assays were performed in duplicate for each sample, and the mean values were used to calculate gene expression levels.

Reverse transcription–quantitative real time PCR was performed to validate the relative expression of the most significant differentially expressed genes in transcriptome microarrays and reference genes (Table S4). This step was performed using a LightCycler^®480 II system (Roche, Basel, Switzerland). Reverse transcription reactions were performed using 500 ng (cells and tissue samples) and 150ng (exosomes samples) of total RNA, random hexanucleotides, and the High-Capacity cDNA (complementary DNA) Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA), following the manufacturer’s instructions. RT-qPCR was performed with assays based on hydrolysis probes using 1 μL of cDNA, 2.5 μL of TaqMan Gene Expression Master Mix and 0.25 μL of TaqMan Gene Expression Assay (Applied Biosystems, Waltham, MA, USA) to a 5 μL final reaction volume. To calculate the efficiency, random-primed qPCR Human Reference cDNA (Takara Bio, Mountain View, CA, USA) was used. ACTB, GUSB, and CDKN1B were selected as endogenous controls using GeNorm software (https://genorm.cmgg.be/ accessed on 9 July 2015) for tissue analysis, whereas ACTB and GAPDH were selected as endogenous controls for exosome samples. Relative gene expression levels were expressed as the ratio of target gene expression to the geometric mean of the endogenous gene expressions, according to the Pfaffl formula [33 (link)].