Computational protocol: Expression analysis of MND1/GAJ, SPATA22, GAPDHS and ACR genes in testicular biopsies from non-obstructive azoospermia (NOA) patients

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Protocol publication

[…] Reverse transcription and subsequent RT-qPCR was performed as previously described []. Briefly, prior to reverse transcription, purified RNA was treated with RNAse-free DNAse 1 (Fermentas, Burlington, Canada) for 40 min. Template cDNA was synthesized from 1 μg of total testicular RNA using SuperScript® III Reverse Transcriptase (Invitrogen, Life Technologies, Carlsbad CA) or RevertAid™ Reverse Transcriptase (Fermentas, Burlington, Canada) with combination of random hexamer and poly(dT) primers (1:1) in a Touchgene Gradient Thermal Cycler (Techne, Burlington, USA). The qPCR conditions were: initial denaturation for 15 min, followed by 40 cycles of denaturation at 94°C for 20 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec. Following PCR reaction, the melting curve was constructed by increasing the temperature from 72 to 95°C to ensure that the correct product is amplified in the reaction. PCR was repeated three times in doublets for each gene, and the average Ct was used for further analysis. Gene-specific primers for PPIA, GAPDHS and ACR were designed using the advantages of Primer3 software and BLAST alignment of Primer-BLAST service from NCBI []. Three best primer pairs overlapping the intron sequence were ordered and after pretesting, the best of them was used for gene expression analysis. Due to the large amount of pseudogenes for GAPDH gene in human genome [], neither of the three primer pairs ordered was suitable because of the dimer formations, and the primers used were as in Barber et al. []. Primers for MND1 and SPATA22 were as in Okada et al. []. The PPIA (peptidylprolyl isomerase A (cyclophilin A)) gene was used as a reference gene. Primer properties are summarized in Table . [...] Experimental data were analysed using STATISTICA 6.0. and GraphPad Prism 5.04. The differences between the control and experimental groups in the relative gene expression were analysed by KW ANOVA, and post hoc analysis was performed by Dunn‘s test. The p value that was equal to or lower than 0.05 was considered to be significant and was indicated with red asterisk in the column. […]

Pipeline specifications

Software tools Primer3, Primer-BLAST, Statistica
Applications Miscellaneous, qPCR
Organisms Homo sapiens
Diseases Heart Arrest, Oligospermia, Azoospermia, Sertoli Cell-Only Syndrome