Quantitative PCR Analysis of Endometrial, Testicular, and Conceptus Transcripts

Real-time RT-PCR experiments were carried out under the consideration of the MIQE guidelines [4 (link)]. RNA samples (2 μg/reaction for endometrial and testicular specimens, and 200 ng/reaction for conceptus specimens) were treated with RNase-free DNase I (Ambion, Austin, TX) for 15 min at 37°C, heat denatured (75°C for 10 min), then reverse transcribed using High Capacity cDNA Reverse Transcription Kit and random hexamers (Applied Biosystems, Foster City, CA). cDNA was purified using the QIAquick^® PCR Purification Kit (Qiagen, Germantown, MD) and cDNA concentration was determined via spectrophotometry. Purified cDNA (50 ng) was used for each PCR reaction.
For endometrial specimen, the mRNA expression of four putative reference genes, glyceraldehyde 3-phosphate dehydrogenase (GAPDHP), 18S rRNA (18S), beta-2-microglobulin (B2M), and beta actin (ACTB) and one non-reference gene, solute carrier family 36 member 2 (SLC36A2) were measured by real-time RT-PCR. For testicular samples, the mRNA expression of GAPDH, 18S, B2M, ACTB, Succinate dehydrogenase complex (SDHA), and beta glucoronidase (GUSB) as putative internal control genes and aromatase (Cyp19a1) as non-reference transcript were determined. For conceptus tissue, the expression of GAPDH, 18S, B2M, ACTB, SDHA, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHAZ) as reference gene candidates and Cyp19a1as non-reference genes was assessed using quantitative PCR. Primers specific for the selected transcripts were designed using Jellyfish 3.3.1 (Field Scientific LLC, Lewisburg, PA) and are listed in Table 1. Specificity of the primers was confirmed via sequencing of the PCR products to confirm amplification of the intended target sequence. Primer efficiency was assessed using Linreg http://www.gene-quantification.de to ensure all primers resulted in PCR efficiencies of at least 1.9. Real-time PCR was completed using SYBR Green PCR Master Mix (Applied Biosystems) with the following cycling conditions: 95°C for 10 min; 40 cycles of 95°C for 15 sec, 59°C for 1 min; 55 to 95°C for dissociation. Each PCR was performed in triplicate. Specificity of amplification was monitored by including non-reverse transcribed RNA reactions for each sample and by completing a dissociation analysis at the end of each real-time run to verify the amplification of a single product. Cycle threshold (Ct) values were obtained through the auto Ct function.