7500 fast qpcr system

Liver stage parasite burden was quantified by qPCR for Pb 18S ribosomal RNA (18S rRNA) as described previously (37 (link)). Total RNA was extracted from liver samples using Trizol according to manufacturer’s instructions. cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). The qPCR reaction samples contained the following reagents in 25 µl volume: 12.5 µl of SYBR Green PCR Master Mix (Applied Biosystems), 0.1 µM of either Pb 18S rRNA primers (forward—5′-AAGCATTAAATAAAGCGAATACATCCTTAC-3′; reverse—5′-GGAGATTGGTTTTGACGTTTATGTG-3′) or mouse β-actin gene (forward—5′-GGCTGTATTCCCCTCCAT-3′; reverse—5′-CCAGTTGGTAACAATGCAAT-3′) and 2 µl of 1:10 dilution of cDNA sample. The reaction was run on 7500 Fast qPCR System (Applied Biosystems) using the following conditions: 15 min at 95°C, 40 cycles with 95°C for 20 s; 60°C for 30 s, and 72°C for 50 s.
cDNA standards were prepared as 10-fold serial dilutions of purified PCR products for both 18S rRNA and β-actin from 10⁸ to 10⁵ copies per 2 µl. Each reaction was set up in triplicate. Livers of naive mice served as a negative control. Parasite load was calculated as ratio of 18S rRNA to host β-actin (housekeeping gene) expression. Protection was defined as a statistically significant reduction of parasite burden in the livers of experimental mice compared with mice immunized with EV.

PMAxx -PCR is an innovative technology that allows differentiation between live and dead microorganisms, based on the loss of cell membrane integrity in dead cells³¹ . This system uses a DNA-intercalating dye, PMAxx, that disrupts DNA transcription only in dead cells, as their damaged cell membranes permit entry of the dye. After photoactivation with a defined wavelength, PMAxx intercalates and binds covalently to DNA. Subsequent amplification of that modified DNA is inhibited, thereby reducing the amplification signal from dead/damaged cells in comparison to that from live cells.
For each qPCR reaction, a 20 μl volume consisting of 10 μl Multiplex Master Mix, 2 μl 10X Exo IPC Mix, 0.4 μl 50X EXO IPC DNA, 1 μl of primer assay, 2 μl of sample/template DNA, and 4.6 μl of sterile deionized water was placed into a 96 well fast plate. Both primers for the multiplex PCR were combined to reach a final concentration representing 5% of the total reaction volume. These qPCR runs were performed on a 7500 Fast qPCR System (Applied Biosystems) under the following conditions: 2 min at 50 °C, then 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C.

Tumor and normal tissue DNA were quantified with a qPCR assay. In brief, a 67 bp. fragment of the APP gene (fwd: 5′‐TCA GGT TGA CGC CGC TGT‐3′ and rev: 5′‐TTC GTA GCC GTT CTG CTG C‐3′, taqman probe: 5′‐FAM‐ACC CCA GAG GAG CGC CAC CTG‐TAMRA‐3′) was amplified (95°C for 20 s denaturation followed by 40 cycles of 95°C for 3 s an 60°C for 20 s) on a 7500 Fast qPCR system (Applied Biosystems). Depending on the C_T‐Value of the qPCR assay the DNA was diluted for the PCR (details are available upon request). The PCR for five monomorphic markers (Primer in Table 1) was performed in two Multiplex‐PCR reactions with following conditions: 95°C denaturation for 5 min, 40 cycles of 95°C for 30 s, 55°C for 45 s, and 72°C for 30 s, and an end‐elongation step at 72°C for 5 min. Capillary electrophoresis was carried out on a Beckman CEQ 800 instrument (Beckman Coulter). Analysis of monomorphic markers was carried out by visual inspection of experienced molecular pathologists comparing the patterns in tumor and adjacent normal tissue DNA.