Computational protocol: The Use of Laser Microdissection in the Identification of Suitable Reference Genes for Normalization of Quantitative Real-Time PCR in Human FFPE Epithelial Ovarian Tissue Samples

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Protocol publication

[…] Reference genes (n = 12) frequently used in the last three years in ovarian cancer research were selected to identify the most stable gene for qPCR normalization. Information regarding the 12 candidate genes is provided in . Primers were generated from published papers or designed using Primer 5 software, and the specificity of each primer was confirmed by primer-BLAST searches. qPCR was performed on an Applied Biosystems StepOnePlus Real-time PCR system (Applied Biosystems, Foster City, CA, USA) using SYBR Green Real-time PCR Master Mix (Toyobo, Osaka, Japan) according to the manufacturer's recommendations. Negative controls (no template in PCR) were performed for each gene, and a melting curve was constructed for each primer to confirm product specificity.Standard curves of a five-fold dilution series were performed for all candidate genes, and the amplification efficiency and correlation coefficient (R2) were generated based on the slope of each standard curve. The equation used to calculate the efficiency was as follows: E% =  [10(−1/slope)–1]%. The delta-Ct method was used to calculate the relative quantification (Q) of the amplification of the candidate genes according to the following formula: Q = E−(sampleCt–minCt), where minCt =  the lowest Ct value observed in a group analyzed for a given primer. [...] Statistical analysis was carried out with SPSS 13.0 statistic software (SPSS, Chicago, IL, USA). Differences were appropriately analyzed by one-way analysis of variance (ANOVA) or a nonparametric (Kruskal-Wallis) test. P<0.05 was considered statistically significant. For the stability analysis of the candidate reference genes, two freely available software programs were applied: NormFinder ( and geNorm version 3.5 ( NormFinder, a Microsoft Excel add-in, focuses on calculating a stability value based on intra- and inter-group expression variations for candidate reference genes. A low value represents a low combined variation and, thereby, reveals high expression stability . GeNorm, another Microsoft Excel application, provides a gene stability measure (M) and ranks the tested genes by stepwise exclusion of the gene with the highest M value. In addition, geNorm calculates the pairwise variations, Vn/Vn +1, between each combination of sequential normalization factors (NF), which allows for the identification of the optimal number of reference genes for accurate normalization . […]

Pipeline specifications

Software tools Primer-BLAST, NormFinder, geNorm
Databases MedGen
Application qPCR
Organisms Homo sapiens
Diseases Ovarian Neoplasms