Taqman universal pcr master mix

The total RNA was extracted from liver tissue samples and selected cell lines, and the first complementary strand was generated. Expression level of SVIP and p97/VCP gene products were analyzed by quantitative real-time PCR using an Applied Biosystems 7500 fast Real-Time PCR system from Life Technologies and TaqMan Universal PCR Master Mix from Roche Applied Science. Pairs of genes were analyzed simultaneously, and h18S ribosomal RNA was used as an endogenous control. The specificity of the priming and amplification was verified with a melt curve for each amplicon. The quantitative real-time PCR was performed in duplicates, and results were averaged. Results are presented as the relative quantification (RQ) as determined by the 2^−ΔΔCt equation.

HSPA1A, HSPA1L and HSPA1B genetic polymorphisms rs1043618, rs2227956, rs2075800 and rs2763979 were selected as the target SNPs, which were referenced from earlier studies [10 (link), 12 (link)–14 (link)]. Blood samples were obtained from all the participants with written informed consent. Each specimen was collected in an ethylenediaminetetraacetic acid (EDTA) tube and centrifuged (2000 g, 20 min). The buffy coat was isolated, and DNA was extracted using a commercial DNA extraction kit (Gentra Corp., Minneapolis, Minn, USA). Genotypes for the selected polymorphisms were screened with ABI TaqMan SNP genotyping assays (Applied Biosystems, Foster City, Calif., USA). The extracted DNA and genotyping assays were added to TaqMan universal PCR master mix (Roche, Branchburg, N.J., USA) according to the manufacturer’s instructions. The genotyping procedures were then performed by using the ABI PRISM 7500 real-time PCR system (Applied Biosystems). The results were analyzed using ABI 7500 System sequence detection software version 2.3 (Applied Biosystems).

Biological specimens for diagnosis of leptospirosis were sampled at admittance of patients. Leptospires in plasma or urine were detected by quantitative real-time PCR (qPCR) using the Light cycler LC480^® system (Roche), and TaqMan^® Universal PCR Mastermix with primers and probe specific for 23S rRNA gene of Leptospira as detailed before [21 (link)]. For quantification of bacterial burden in plasma by PCR, serial dilutions of genomic DNA extracts from L. interrogans serogroup Icterohaemorragiae serovar Copenhageni were performed. These dilutions corresponded to concentrations from 4 x 10⁶ to 4 bacteria/ml and the number of bacteria per ml in plasma samples was inferred from the cycle threshold (Ct) values of PCR according to the log-transformed standard curve, as detailed in previous reports [22 (link)].

After the last day of the PCMS protocol, mice were euthanized by rapid cervical dislocation. Instantaneously after decapitation, tissues used for molecular analysis were snap-frozen in liquid nitrogen and kept at  − 80 °C. Adhering to Takara’s RNA isolation protocol (RNAisoPlus; Takara Bio, Shiga, Japan), total RNA of FCX, thalamus, hypothalamus, hippocampus, TG, and DRG were isolated. A high-capacity cDNA transcription kit (Applied Biosystems, Foster, CA, USA) was used to generate cDNA from each tissue RNA sample. Gene-specific and predesigned TaqMan primer sets and probes (Applied Biosystems, Foster City, CA) were used to ascertain TNF-α and IL-6 gene expression. The Applied Biosystems 7900HT real-time RT-PCR system and TaqMan Universal PCR Master Mix (Roche Applied Science, Mannheim, Germany) were used to perform real-time RT-PCR corresponding to the guidelines of the manufacturer. Normalization of the values to those of housekeeping gene beta-actin was done for endogenous quality control. Attributable to the small size of some tissues or low concentration of mRNA, the RT-PCR results in some cases yielded no data value. In these instances, the values cannot be considered for inclusion in the final statistical analysis.