Iq multiplex powermix

Tick pool DNA was screened by multiplex for bacterial presence using a multiplex for R. rickettsii, R. parkeri, Rickettsia amblyommatis, Ehrlichia ewingii, and Ehrlichia chaffeensis using iQ Multiplex Powermix (Bio-rad, Hercules, CA) in 20 μL reactions with 2 μL of DNA extract [39 (link)] (Table 1). Plasmids containing a single copy of each targeted gene were employed as positive controls.
Positives identified by the multiplex screening assay were confirmed by additional sequencing of rickettsial htrA (17 kDa) 17k3 and 17 k5, Sca0 (ompA); 190.70p and 190.602n, Sca5 (ompB);120-M59 and 120–807 and/or 120–2788 and 120–3599, or 23S-5S IGS; RCK/23-5N1F and RCK/23-5N1R (Table 1) [44 (link)–46 (link)]. DNA extracted from R. sibirica culture was utilized as a positive control. Reaction mixes for PCR were prepared in 25 μL reactions using 5PRIME HotMasterMix (Quantabio, Beverly, MA) and 800 μg/mL bovine serum albumin (BSA) water [47 (link)]. Previously published PCR thermal cycling conditions were used (Table 1) and resultant products were separated and visualized on an 1.5% agarose gel stained with ethidium bromide.

In reverse transcription (RT) reactions, 0.5 μg of total RNA were used as template. Reverse transcription was performed using iScript cDNA Synthesis kit according to manufacturer’s instructions (Biorad). For each sample, both RT⁺ and RT^– reactions were run by adding or lacking reverse transcriptase enzyme. Quantitative real-time PCR (qPCR) was performed using CFX96 Touch™ Real-Time PCR Detection System (Biorad) machine using iQ™ Multiplex Powermix (Biorad). qPCR experiments for L1 elements were performed in duplex using FAM-labeled Taqman probes against the specific 5′UTR L1 subfamily together with VIC-labeled Taqman probe against housekeeping gene UbC. PCR amplification was performed in a final volume of 20 μL using the following parameters: (1) 95°C for 20 s, (2) 95°C for 10 s, (3) 59°C for 30 s. Steps (2) and (3) were repeated 40 times. In qPCR experiments, we loaded both RT⁺ and RT^– samples to control residual genomic DNA contamination. We accepted a minimum of 6 cycles of difference between RT^– and RT⁺ samples for the 5′UTR target sequence. For relative quantification of the transcripts, the 2^(–ΔΔCt) method was used (Livak and Schmittgen, 2001 (link)). For each assay, at least three independent qPCR technical replicas were performed on all samples. Primers and probes used are listed in Supplementary Table 5.

Eimeria species were identified by qPCR using specific primers targeting SCAR markers derived from RAPD fragments for E. acervulina, E. tenella, and E. necatrix, and microneme protein gene 1 for E. maxima [19 (link)]. Oocysts from positive fecal samples were isolated, purified, and concentrated with a saturated salt solution [20 ]. Then, oocysts were washed three times with distilled water by repeated centrifugation [21 (link)]. Genomic DNA was extracted from 0.25 mg fecal sample and 100 μL oocysts pellets using the Isolate Fecal DNA (Bioline) kit according to the manufacturer’s instructions. The qPCR reactions were performed in the CFX96 Touch™ Real-Time PCR detection system (Bio-Rad, London, UK) using IQ Multiplex Powermix (Bio-Rad, London, UK) in a final volume of 20 µL. The amplification conditions were similar to the conditions described by Blake et al. [19 (link)] as follows: 1 × initial denaturation at 95 °C for 20 s; 40 × 95 °C denaturation, 15 s, and primer hybridization combined with 60 °C extension, 30 s. Data were collected at the end of each cycle.

Pellets of approximately 10 × 10⁶ cells were collected from sub-confluent T175 cm² flasks. Total RNA and DNA were isolated using an RNA/DNA isolation kit (Qiagen, Germantown, MD, USA) and measured using a NanoDrop (Thermo Fisher). From RNA, complementary DNA (cDNA) was made using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). Quantitative real-time PCR was run for single genes (EvaGreen Power mix, Bio-Rad, Hercules, CA, USA) or in a multiplex set (iQ Multiplex Power mix, Bio-Rad) in a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Bio-Rad CFX Manager software calculated starting quantities for samples, based on a standard curve with known starting quantities. All values were normalized to endogenous reference genes based on geNorm (BioGazelle, Technologiepark 3 Zwijnaarde, 9052, Belgium) analysis to find the genes with the highest expression stability. For gene expression, we used the endogenous reference genes for glyceraldehyde 3-phosphate dehydrogenase and 14-3-3-zeta. We used 14-3-3-zeta and beta-2-microglobulin as the endogenous reference genes for DNA copy number studies. Student’s t-tests were used to analyze data (Prism, GraphPad, San Diego, CA, USA).