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Figure 1 | BMC Medical Genetics

Figure 1

From: Evidence for population variation in TSC1 and TSC2 gene expression

Figure 1

Method for correcting genomic and cDNA allele ratios (AR). Genomic DNA segments containing marker SNPs are amplified by PCR and used as templates in SNaPshot primer-extension assays. Extended primers containing one of two different fluorescently labeled nucleotides at their 3'-ends are resolved by capillary electrophoresis and the ratio of peak heights calculated. An average genomic AR for a specific SNP is determined from all the genomic samples (each analyzed in triplicate). A correction factor (CF) is then calculated as the inverse of the average genomic AR. The genomic samples analyzed in triplicate are then individually multiplied by the CF to normalize the data to approximately 1.0, which is the theoretical ratio of two gene alleles in genomic DNA. The corrected average genomic AR is then determined. For each RNA sample, cDNA is synthesized and heterozygous SNP containing segments are amplified by PCR in triplicate and subjected to a SNaPshot PCR reaction. Samples are run on a capillary sequencer and the ratio of heterozygous peak heights is determined. Individual cDNA ARs are calculated and corrected by multiplying by the CF. The average corrected cDNA AR for each sample is then calculated. A sample is designated as showing AEI if the average corrected genomic AR and average corrected cDNA AR differ by greater than 2X the standard error of the mean and by at least 10%.

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