Computational protocol: Reference genes for normalization of gene expression studies in human osteoarthritic articular cartilage

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Protocol publication

[…] RNA was reverse transcribed to cDNA using Transcriptor Reverse Transcriptase (Roche Applied Science, Barcelone, Spain) and random hexamers in a total volume of 20 μl according to the manufacturer's instructions. Limited RNA quantities dictated input RNA amounts to be 220 ng. Complementary DNA was stored at -20°C until 0.5 μl were used in each downstream PCR. Oligonuleotide primers were designed using Primer3 software [] in different exons to help prevent amplification of genomic DNA. PCRs were performed using DyNAmo™ SYBR® Green qPCR kit (Finnzymes, Espoo, Finland) and Chromo4™ real-time PCR detection system (MJ Research, Basel, Switzerland). The PCR reaction volume was 10 μl with 0.1 μM of the primers. Cycle conditions were set as an initial denaturation step for 10 min at 95°C, followed by 40 cycles of 10 s at 94°C for template denaturation, 15 s for annealing and 10 s at 72°C for extension. Table shows the annealing temperatures and the MgCl2 concentrations specific for each set of primers. All reactions were run in duplicate and all samples were analyzed in the same run to exclude between-run variations. Each RNA sample was controlled for genomic DNA contamination for each gene-specific PCR by a reaction well without reverse transcription. Reagent contamination was also examined by a reaction mix without template. Specificity of the PCR reactions was confirmed by melting curve analysis of the products as well as by size verification by DNA electrophoresis in agarose gels. [...] PCR efficiency was calculated with the LinRegPCR program [] from raw fluorescence data taken from the Chromo4™ real-time PCR detection system. According with this method PCR efficiency is the slope of the straight line that best fit the log-linear part of the amplification curve. Mean efficiencies were determined in sample duplicates and used to adjust Ct values. Ct values, the cycle number at which the fluorescence signal of the sample exceeds background fluorescence, were used for the quantitative comparison of the amplification rates. They were obtained using Opticon Monitor™ version 3.0 software, provided with the Chromo4™ real-time PCR detection system. After baseline subtraction, threshold lines were manually established for each gene to cross the the log-linear part of the fluorescence curves. Mean Ct values of the duplicates were determined and transformed into relative quantities. [...] Mann-Whitney U tests were performed with Statistica, version 7 (Statsoft, Tulsa OK). The softwares geNorm™, version 3.4 [] and NormFinder [] were used to calculate stability of the candidate reference genes. The first, geNorm, relies on the principle that the expression ratio of two reference genes should be identical in all samples, regardless of the experimental condition. It calculates the expression stability measure (M) for the set of candidate reference genes and by stepwise exclusion of the least stable gene in each step arrives to the the most stable pair of reference genes. It provides also a way to estimate the best number of required reference genes. NormFinder follows a different approach: it calculates a stability value for each individual candidate reference gene taking into account separation of samples in the different groups that are of interest in the specific area of research []. In this case, the stability value is based on the combined estimate of intra- and intergroup variation of gene expression. […]

Pipeline specifications

Software tools Primer3, LinRegPCR, Statistica, NormFinder
Applications Miscellaneous, qPCR
Organisms Homo sapiens
Diseases Cartilage Diseases, Osteoarthritis, Osteoarthritis, Hip