Computational protocol: Expression of proto-oncogene KIT is up-regulated in subset of human meningiomas

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Protocol publication

[…] Primers for the genes of interest (GI) and reference (GR) were designed using Primer Express 3.0 software (ABI). Specificity of the primers was confirmed by blastn algorithm of the BLAST program. GAPDH was used as an endogenous control or GR, as it showed stable expression in both control and test samples. SYBR® green (ABI) assays were performed for relative quantification of KIT and KITLG transcripts on 7500 Real-Time PCR System (ABI). Commercially purchased total RNA of human ♂ meninges and ♀ dura were used as controls or calibrators (BioChain and Clontech, Mountain View, CA, USA; respectively). Two fold dilution series of the cDNA template were assayed to generate standard and disassociation curves. Standard curves of primers, for all the three genes, had slope values within the range of −3.3 to −3.4, R2 >0.99 (co-efficient of determination) confirming comparable PCR efficiencies and good fit of data points, respectively. Further single melting curve peaks validated specific amplification (data not shown). Standard curve was employed to ascertain the amount of sample cDNA to be used for RT-qPCR. All the assays were performed using respective gene’s 100 nM of forward and reverse primers, in a final reaction volume of 20 μl. Universal cycling conditions as recommended by ABI were employed to amplify GIs and GR in separate wells. The results were ratified, when of the triplicate Ct values (Cycle threshold), at least two were concordant. Expression levels were calculated by the relative quantification (RQ = 2−ΔΔCt) method []. ΔΔCt is the cycle threshold normalized first with the endogenous control (ΔCt = Ct GI - Ct GR) and then with the calibrator sample (ΔΔCt = ΔCt Sample - ΔCt Calibrator). SDS 7500™ Software v2.0.3 was used to analyze the data and heat map was generated using Data Assist™ Software v2.0 (ABI). [...] Based on relevant literature review, KIT exons were selected for mutational screening. PCR was performed with 50 ng of tumor and matched blood DNA as template from the KIT positive cases in a reaction volume of 35 μl, using primers specific for exons 1, 9, 10, 11, 12, 13 and 17 of KIT. Commercially purchased human brain (BioChain) and blood DNA from a healthy volunteer were used as controls. The amplicons were electrophoresed on a 1% agarose gel and purified using QIAquick gel extraction kit as per manufacturer’s guidelines (Qiagen). Sequencing (2X) was conducted with these purified PCR fragments as templates using Big Dye® Terminator v3.1 chemistry (ABI) and standard protocol, on 3130xl Genetic Analyser (ABI). Sequences were screened for mutations and compared with corresponding human reference KIT exon sequence at NCBI, using Sequence Analysis software v5.3.1 and SeqScape® software v2.6 (ABI). […]

Pipeline specifications

Software tools Primer Express, BLASTN, SeqScape
Applications Sanger sequencing, qPCR
Organisms Homo sapiens
Diseases Meningioma, Neoplasms