Iscript one step rt pcr kit

S. mutans was cultured in BHI supplemented with peptides or the vehicle. Cultures were centrifuged, and total RNA was isolated and purified from the bacterial pellets using the GRS Total RNA Kit—Bacteria (GK16.0100; GRISP Research Solution, Porto, Portugal), following the manufacturer’s instructions. Purified RNA was subjected to DNase I treatment to remove contaminating DNA. The RNA yield and purity were assessed by measuring the absorbance, and only samples with a ratio of 260/280 nm in the 1.8–2 range were used. cDNA was generated using the cDNA RT kit with an RNAse inhibitor (Applied Biosystems; Monza, Milan). Quantitative PCR (qRT-PCR) was performed using SYBR green mixture (iScript One Step RT-PCR kit; Bio-Rad, Segrate, Milan) to determine the transcript levels of genes using the oligonucleotides listed in Table 3. The gyrA gene served as the housekeeping gene.

Gene expression of RNA used for microarray analysis was measured by q-PCR on an iCyclerIQ (Bio-Rad Laboratories, Richmond, CA) using an iScript One-Step RT-PCR kit with SYBR Green (Bio-Rad Laboratories, Richmond, CA). Reaction parameters were as follows: cDNA synthesis at 50°C for 20 min, transcriptase inactivation at 95°C for 5 min, PCR cycling at 95°C for 10 sec, and 60°C for 30 sec for 40 cycles. The following primers were used for RT-PCR: β-actin 5’-AGAAGGAGATCACTGCCCTG-3′ (forward), 5’-CACATCTGCTGGAAGGTGGA-3′ (reverse); CHI31 5’-GTGAAGGCGTCTCAAACAGG-3’ (forward), 5’-GAAGCGGTCAAGGGCATCT-3’ (reverse); TRADD 5’-GCTGTTTGAGTTGCATCCTAGC-3’ (forward), 5’-CCGCACTTCAGATTTCGCA-3’ (reverse); NF1 5’-AGATGAAACGATGCTGGTCAAA-3’ (forward), 5-CCTGTAACCTGGTAGAAATGCGA-3’ (reverse); RelB 5’-CAGCCTCGTGGGGAAAGAC-3’ (forward), 5’-GCCCAGGTTGTTAAAACTGTGC-3’ (reverse); CASP4 5’-TTTCTGCTCTTCAACGCCACA-3’ (forward), 5’-AGCTTTGGCCCTTGGAGTTTC-3’ (reverse); FGFR3 5’-TGCGTCGTGGAGAACAAGTTT-3’ (forward), 5’-GCACGGTAACGTAGGGTGTG-3’ (reverse); PDGFA 5’-GCAAGACCAGGACGGTCATTT-3’ (forward), 5’-GGCACTTGACACTGCTCGT-3’ (reverse); EGFR 5’-CTACGGGCCAGGAAATGAGAG-3’ (forward), 5’-TGACGGCAGAAGAGAAGGGA-3’ (reverse); AKT2 5’-ACCACAGTCATCGAGAGGACC-3’ (forward), 5’-GGAGCCACACTTGTAGTCCA-3’ (reverse); Nestin 5’-GGCGCACCTCAAGATGTCC-3’ (forward), 5’- CTTGGGGTCCTGAAAGCTG-3’ (reverse).

Dengue virus serotyping was carried out in a fourplex Taqman Real-Time RT-PCR detection platform as described by Johnson et al. (2005) [10 (link)]. PCR reactions were prepared in a cocktail of 12.5 μl of 2X RT (reverse transcriptase)-PCR Mix (i-Script One Step RT-PCR kit, Biorad, USA), 0.5 μl of each primers (DENV 1 and DENV 3 primers: 50 μM; DENV 2 and DENV 4 primers: 25 μM), 0.45 μl of each probes (10μM), 0.5 μl of RT Enzyme Mix, and 1.2 μl of nuclease-free water. Positive controls for each serotype comprised of RNA from previously confirmed dengue patients, and obtained from the Virology Unit, IMR. The negative control consisted of reactions without an RNA template, which was substituted with 5μl of nuclease-free water. Taqman Real-Time RT-PCR amplification was performed on the CFX 96 (Biorad, USA) platform at 50°C for 10min, 95°C for 5min, followed by 45 cycles of 95°C for 15 sec and 60°C for 30 sec. The aforementioned PCR mix and cycling conditions were optimized by the Virology Unit. io: dx.doi.org/10.17504/protocols.io.rabd2an.[PROTOCOL DOI]

Levels of HIV-1 RNA in plasma of infected BLT mice were determined by a RT-PCR assay. Plasma was separated from peripheral blood and stored at −80°C until use. Viral RNA was isolated with a QIAamp viral RNA mini kit (QIAGEN). The RNA was eluted in 25 µl of RNase-free water and 5 µl of elution was applied for qRT-PCR using iScript One-step RT-PCR kit (Bio-Rad laboratories) with following primers/probe specific to HIV-1_NFNSX Gag region. Primer Sequence 1: 5’-CCCTACCAGCATTCTGGACATAAG-3’, Primer Sequence 2: 5’-GCTTGCTCGGCTCTTAGAGTT-3’ Probe: 5'-FAM-ACAAGGACCAAAGGAACCCTT-BHQ1-3'. With these primers/probes, HIV-1 RNA can be quantitatively detected within a range of 10³ copies to 10⁸ copies/ml.

The real-time RT-PCR was conducted using the iScript™ One-Step RT-PCR kit (BioRad, Hercules, CA, USA) with the Rotor-Gene Q Real Time PCR machine (Qiagen, Germantown, MD, USA). The samples were assayed in a 25 µL reaction containing 2 µmol/L of forward primer (CHIK/E1/10367/+: 5′-CTCATACCGCATCCGCATCAG-3′), 2 µmol/L of reverse primer (CHIK/E1/10495/-: 5′-ACATTGGCCCCACAATGAATTTG-3′), 5 µL of extracted RNA, 0.25 µL of RNA transcriptase, and 12.5 µL of SYBR^® Green Premix. The concentrations of Taq polymerase, buffer, dNTPs, and Mg²⁺ used were based on the recommendations of the manufacturer. The RT-PCR thermal cycling condition comprised 30 min of reverse transcription step at 50 °C, 15 min of initial denaturation at 95 °C, followed by 40 cycles of amplification steps of denaturation at 95 °C for 30 s, annealing at 55.8 °C for 45 s, extension at 72 °C for 60 s, and a final extension at 72 °C for 10 min. A melting curve was generated after the amplification step at 70–99 °C. A standard curve was constructed by using the synthesized RNA standard with copy numbers ranging from 10⁰ to 10¹⁰.