Faststart universal probe master

Total RNA was extracted from cells using RNAiso Plus reagent (TaKaRa Bio, Shiga, Japan). One microgram of total cellular RNA was converted to cDNA using the PrimeScript 1st-Strand cDNA Synthesis Kit (Takara Bio). cDNA (1 μL) was amplified with the FastStart Universal Probe Master (Roche Life Science) using a 7500 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). The PCR conditions were 50°C for 2 min and 95°C for 15 s, followed by 45 cycles at 95°C for 15 s and 60°C for 1 min. The primer sequences and fluorescent probes were as follows: IL-6, forward 5′-GCC CAG CTA TGA ACT CCT TCT-3′, reverse 5′-CTT CTC CTG GGG GTA CTG G-3′, Universal Probe #68 (Roche Life Science); 18S, forward 5′-AAA TCA GTT ATG GTT CCT TTG GTC-3′, reverse 5′-GCT CTA GAA TTA CCA CAG TTA TCC AA-3′, Universal Probe #55 (Roche Life Science). The expression levels of IL-6 mRNA were calculated as the ratio of its value to that of 18S rRNA as an internal control.

Huh7.5.1 cells were transfected with various miRNA mimics or siRNAs for 72 h and then harvested. Total cellular RNA was prepared using the RNeasy Mini Kit. RNA quality and quantity were assessed on a Nanodrop spectrophotometer. Complementary DNA (cDNA) was then synthesized from total cellular RNA using First-Strand cDNA Synthesis Kit (Roche). The mRNA levels of target genes were subsequently determined by qPCR using gene-specific primers and probes (IDT) and FastStart Universal Probe Master (Roche) on an ABI ViiA 7 Real-Time PCR System. Relative mRNA levels were calculated using the ΔΔCT method, with 18S rRNA or GAPDH RNA (Applied Biosystems) as internal control for normalization.

A Real-Time PCR flowchart for identification of T. cruzi DTUs in biological samples using TaqMan probes (MTq-PCR) is shown in Fig 1. Oligonucleotide concentration and sequence information is detailed in Table 2. TaqMan probes were purchased from Integrated DNA Technologies, Inc. (USA). SL-IR and 18S-COII MTq-PCR assays were carried out using 1X QIAGEN Multiplex PCR Kit (QIAGEN, USA), while the 24Sα-III/IV MTq-PCR used 1X FastStart Universal Probe Master (Roche, Germany). All PCR reactions were carried out with 2 μL of resuspended DNA in a final volume of 20 μL. Optimal cycling conditions for the SL-IR and 18S-COII MTq-PCR assays were initially 15 min at 95°C followed by 40 cycles at 95°C for 30 sec and 60°C for 1 min in an Applied Biosystems (ABI 7500, USA) device. In turn, optimal cycling condition for the 24Sα-III/IV reaction was an initial cycle of 10 min at 95°C followed by 40 cycles at 95°C for 30 sec and 57°C for 1 min in a Rotor-Gene 6000 (Corbett, UK) device.

HEK-Ds1-mCherry cells were grown for 24 hr. Doxycycline (DOX) was added for 0, 0.5, 1 and 2 hr. RNA was isolated with TRIzol reagent (Life Technologies). 1 µg of RNA was reversed transcribed (SuperScript III, Termo Fisher Scientific). mRNA expression was evaluated with the TaqMan Gene Expression Assay (Applied Biosystems) using the FastStart Universal Probe Master (Roche). Relative expression of the mRNAs was normalized to GAPDH. Real-time PCR was performed in triplicate.
Primers sequence:
For mCherry: Forward: AGGACGGCGAGTTCATCT, Reverse: CCCATFGTC TTCTTCTGCATTA
For GAPDH: Forward: GCTGGCATTGCCCTCAAC, Reverse: CATGAGGTCCAC CACCCTG

qRT-PCR was performed with a FastStart Universal Probe Master (Roche Applied Science, Mannheim, Germany) and analyzed with a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), following the manufacturer’s instructions. Relative transcript abundance was normalized for that of Hprt mRNA. The information for the TaqMan primer/probe (Applied Biosystems, Carlsbad, CA, USA) used for real-time PCR is listed in Supplementary Table S2.