Abi prism 7900 sds software

The RNA isolated from the breast cancerous tissues and paired non-cancerous tissues were kept using TRIzol^® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. β-actin mRNA was the reference gene used as the internal control. The primers of BRCA1 and β-actin (Invitrogen Life Technologies) are shown in Table I. The PCR cycle conditions used are 95°C for 2 min; 40 cycles at 95°C for 10 sec, 60°C for 30 sec, and 70°C for 30 sec; and final extension at 72°C for 7 min. Dissociation curve analyses were used to confirm the specificity of the SYBR^® Green (Invitrogen Life Technologies) signals in each experiment. Data were analyzed using ABI Prism 7900 SDS software (Applied Biosystems, Waltham, MA, USA). The mRNA expression of BRCA1 was analyzed using the 2^−ΔΔCt method (26 (link)). Fluorescent data were converted into RQ measurements, which stand for relative expression automated by the system software. Thermal dissociation plots were examined for biphasic melting curves. To ensure experiment accuracy, quantitative PCR products were randomly selected for sequencing.

For determining the APOE epsilon alleles ε2, ε3, and ε4, manufacturer protocol from Applied Biosystems TaqMan SNP Genotyping Assays C___3084793 and C____904973 (Thermo Fisher Scientific) was followed. C___3084793 and C____904973 identify the single-nucleotide polymorphisms (SNP) rs429358 and rs7412, respectively. Briefly, the T allele at rs429358 and the C allele at rs7412 indicate the ɛ3 allele, whereas the T allele at both SNPs identify the ɛ2 allele, and the C allele at both positions determine the ɛ4 allele. PCR amplification reactions were performed in a final 10 µl volume containing 100 ng genomic DNA, TaqmanTM Genotyping Master Mix (2X), and TaqmanTM SNP Genotyping Assay (50X). The reaction was incubated at 50  ${}^{\circ }$ C for 2 min, followed by 95 °C for 10 min, and 40 cycles at 95 °C for 15 s and 64 °C (rs429358) or 61 °C (rs7412) for 1 min. The fluorescent signal generated by PCR amplification is detected by ABI PRISM Sequence Detection Systems 7900 (Applied Biosystems) and the allelic discrimination analysis is performed by ABI Prism 7900 SDS Software (Applied Biosystems).

ABI PRISM 7900 SDS software (version 2.4; Applied Biosystems) was used to evaluate the amplification and dissociation curves and determine the Ct values. Statistical analyses were performed in Prism 6.0 (GraphPad). Parasite densities (parasites/μl) and starting copy number of P. falciparum 18S rRNA plasmid from the standards were plotted against Ct values derived from the nested qPCR assay to generate standard curves. For all samples with “unknown” parasite concentrations, parasite densities were estimated from a regression line fit to the linear part of the standard curves using the average of nested qPCR-derived Ct values (performed in duplicate for all samples). The strength and significance of correlations were assessed with the Pearson’s correlation coefficient.