Realplex software

Total RNA was isolated from 10⁶ ATII cells 24 h after isolation using RNeasy MiniKit (Qiagen, Hilden, Germany). Reverse transcription was performed on 0.8 µg to 1.3 µg total RNA using the SuperScript VILO cDNA synthesis kit according to manufacturer's protocol. The following validated QuantiTect primer assays (Qiagen, Hilden Germany) were used: HMBS, Rn_Hmbs_1_SG; Syt1, Rn_Syt1_2_SG; Syt-2, Rn_Syt2_1_SG; Syt3, Rn_Syt3_1_SG; Syt5, Rn_Syt5_1_SG; Syt6, Rn_Syt6_1_SG; Syt7, Rn_Syt7_1_SG; Syt-9, Rn_RGD:621169_1_SG; Syt10, Rn_Syt10_2_SG; complexin-1, Rn_Cpl1_1_SG; complexin-2, Rn_Cpl2_1_SG; complexin-3, Rn_Cpl3_1_SG; complexin-4, Rn_Cpl4_1_SG. Amplification was performed on a realplex2 mastercycler (Eppendorf, Hamburg, Germany) using the XPress Syber Green ER qRT-PCR super mix. Each reaction was carried out on cDNA from three or more independent isolations (cDNAs were used at 1-, 10- and 100-fold dilutions). Specificity of PCR reactions was confirmed by melting points analysis of PCR products. Realplex software (Eppendorf, Hamburg, Germany) was used for data acquisition and analysis. Correction for PCR performance as well as quantification relative to housekeeping gene HMBS was carried out as described (Pfaffl, 2001 (link)).

Total RNA was extracted from homogenates of the left lung lobe from each mouse using NucleoSpin RNA columns (Macherey-Nagel) according to the manufacturer’s instructions. RNA samples were reverse transcribed using random primers and M-MLV Reverse Transcriptase (SuperScript II, Invitrogene) according to the manufacturer’s instructions. The primers (from Sigma–Aldrich, St. Louis, MO) used are RSV N gene: F-5’-AGATCAACTTCTGTCATCCAGCAA-3’ and R-5’-TTCTGCACATCATAATTAGGAGTATCAAT-3’ and mHPRT gene: F-5’- CAGGCCAGACTTTGTTGGAT-3’ and R-5’-TTGCGCTCATCTTAGGCTTT-3’. Real time PCR was run in triplicate for each gene in 96 well microplates using the MasterCycler^® ep realplex (Eppendorf) and SYBRGreen PCR Master Mix (Eurogentec). Fluorescence curves were analyzed using the Realplex software (Eppendorf) to determine the cycle threshold (Ct) values for each gene. Individual data were normalized to HPRT mRNA, by calculating the ΔCt (median Ct (gene) - median Ct (HPRT)) and the ΔΔCt value (sample ΔCt - mean ΔCt of control group) was used to calculate relative gene expression (2^-ΔCt) or relative quantity using the formula RQ=2^-ΔΔCt.

Extracted RNA (500 ng) was transcribed into complementary DNA (cDNA) using the RT² First Strand Kit. Transcription was performed according to the manufacturer’s protocol. In brief, 10 µL of a reverse transcriptase mixture was added to the RNA samples. The mixture was incubated for 15 min at 42 °C and then for another 5 min at 95 °C. From each resulting cDNA sample, 675 µL were mixed with 675 µL of RT² SYBR Green/ROX qPCR Mastermix. A volume of 25 µL from each sample was transferred into a specially designed RT² Custom Profiler PCR 96-well plate using a TECAN freedom evo (TECAN, Crailsheim, Germany). 96-well plates were pre-spotted with specific primers according to the results of 2D-GE. Plates were sealed with cap s–trips and placed into the Mastercycler 2S (Eppendorf, Hamburg, Germany). The qPCR was carried out with the following PCR program: 10 min at 95 °C followed by 40 cycles of 15 s at 95 °C, and 1 min at 60 °C. At the end of the PCR program, a melting profile of the DNA amplifications was measured with the following settings: 95 °C for 15 s, 60 °C for 15 s and a final temperature gradient from 60 °C to 95 °C over 20 min. PCR data were analyzed with the realplex software from Eppendorf and with an online software from QIAGEN [25 ].

Total RNA was isolated from 10⁶ ATII cells directly after isolation or following 48 h of culture in MucilAir medium with an RNeasy MiniKit (Qiagen, Hilden, Germany). Reverse transcription was performed on 0.8 µg to 1.3 µg total RNA using the SuperScript VILO cDNA synthesis kit according to manufacturer's protocol and validated QuantiTect primer assays (Qiagen, Hilden Germany) Amplification was performed on a realplex2 mastercycler (Eppendorf, Hamburg, Germany) using the XPress Syber Green ER qRT-PCR super mix. Each reaction was carried out on cDNA from ≥three independent isolations (cDNAs were used at 1-, 10- and 100-fold dilutions). Specificity of PCR reactions was confirmed by melting points analysis of PCR products. Realplex software (Eppendorf, Hamburg, Germany) was used for data acquisition and analysis. Correction for PCR performance as well as quantification relative to housekeeping gene Hmbs was carried out as described previously (Pfaffl, 2001 (link)).

On the 15^th day postinfection, samples of lung, spleen, bladder, heart, intestine, and skeletal muscle were obtained from the infected BALB/c mice for the quantification of the parasite load. The fragments were transferred to formaldehyde (10%) and were then processed by gradual dehydration in ethanol solutions, followed by immersion in xylene, and subsequently embedded in paraffin. Tissue sections 5 μm thick were obtained and stained with hematoxylin and eosin (H&E) and analyzed by light microscopy. The number of amastigote nets was counted in 20 random microscope fields using a 400x magnification. In parallel, tissue fragments were submitted for DNA extraction using the DNAeasy Blood and Tissue Kit as recommended by the manufacturer. The tissue parasitic load was also performed using quantitative PCR as previously described [21 (link)]. The cycle threshold values obtained by the Eppendorf RealPlex software were converted to the number of parasites per 5 ng of tissue DNA. Their averages were normalized according to the TNF-α gene.