Icycler iq5

Arabidopsis leaves were infiltrated with 300 nM PpNLP or PccNLP, heat-denatured (1.5 hours, 95°C) proteins or mutant versions (H121A D124A), respectively. RNA was isolated using the RNeasy Plant MiniKit (Qiagen) and synthesis of cDNA was performed by means of the RevertAidTM MuLV reverse transcriptase (Fermentas). Quantitative real-time PCR amplification was carried out in the presence of SYBR Green (Bio-Rad) with an iQ5 iCycler (Bio-Rad). Amplification of EF1-α served as internal standard. Data were analyzed according to the 2^−ΔΔCT-method [44] (link). Gene induction (fold change) by NLPs was presented as the average of 3 determinations plus or minus standard deviation relative to the expression level of H₂O infiltration.

Cell were harvested and homogenized in 1 mL TriPure isolation reagent (Roche, Mannheim, Germany), and total RNA was extracted according to the manufacturer’s protocol and resuspended in RNase-free water. Reverse transcription was performed as previously described [31 (link)]. cDNAs (0.04 µg of cDNA template in 2 µL) were mixed with 10 µM of selected primer pair (Table 1) and Perfecta SYBR green reaction mix (VWR, Radnor, PA, USA) in a final volume of 15 µL. Reactions were performed with an IQ5 iCycler (Bio-Rad, Herts, UK) as previously described [64 (link)]. Annealing temperatures are given in Table 1. Amplification levels were normalized to those of β-actin. All samples were measured in duplicate.

Real-time PCR was conducted using iScript One-Step RT-PCR Kit (Hercules, CA, USA) with SYBR Green in BIO-RAD iQ5 iCycler. Reaction was prepared in a total of 30 µL reaction mix which composed of 15 µL 2× SYBR Green RT-PCR Reaction mix, 200 nmol of forward and reverse primers respectively, 100 ng total RNA, 0.5 µL iScript Reverse Transcriptase for One-Step RT-PCR and ddH₂O was added to a final volume of 30 μL. The following programme was used to amplify the genes: 10 min at 50 °C and 5 min at 95 °C followed by 30 cycles of 95 °C for 10 s and 54 °C for 30 s. Finally, the reaction was completed by the melt curve analysis which consisted of incubation for 1 min at 95 °C, 1 min at 55 °C, and 10 s at 55 °C for 80 cycles, with an increase of 0.5 °C after each cycle.

Thermal denaturation assays were performed on a Bio-Rad IQ5 ICycler. Recombinant proteins (~4 μM) were mixed with Sypro orange (×5) in a 50-μL reaction. Fluorescent readings were taken every 30 seconds between 20 °C and 70 °C. The melting temperature was determined from the maximum of the first derivative of the melting curve, and the mean melting temperature of three replicates was calculated for each protein.

The extracted DNA from bacterial consortia was diluted to 15 ng/uL and qPCR measurements were performed in triplicate in 10-μL reactions in an iQ5 iCycler (Bio-Rad, Veenendaal, the Netherlands) using the iQ SYBR Green Supermix kit (Bio-Rad). Standard curves were obtained using serial dilutions of a known amount of plasmid DNA containing a fragment of the 16S rRNA gene of Dehalococcoides mccartyi CBDB1. The primers were 341 F 5’-CCTACGGGAGGCAGCAG-3’ and 534 R 5’-ATTACCGCGGCTGCTGGC-3’, and the thermal cycling conditions were 95 °C for 10 min, followed by 39 cycles of 95 °C for 15 s, 60 °C for 30 s, 72 °C for 30 s.

Single strand cDNA was generated from isolated total RNA samples using iScript cDNA synthesis kit (Bio-Rad) as per manufacturer instructions. The qRT-PCR reactions were prepared using IQ SYBR Green qRT-PCR kit (Bio-Rad) as per manufacturers instructions with custom designed primer pairs to select gene targets (S2 Table) and run on iQ5-icycler (Bio-Rad). Each primer set was designed to generate 200 bp PCR amplicons except those to PG_RS02100, which produce a 50 bp PCR amplicon and was used to qualitatively verify wildtype expression and loss of expression in the mutant. The qRT-PCR data was analyzed by the standard comparative Ct method [13 (link)]. To determine the fold-change in expression value for each target gene, the 2^-ΔΔCt was calculated. If the fold-change value was less than 1, indicating reduction in expression in the mutant relative to the wildtype, the negative inverse of the fold-change value was calculated. A change in relative expression of 3-fold or greater was selected as the threshold in our analysis as a notable difference in expression of a target gene in the mutant W83Δ514 relative to wildtype W83. Changes below 2-fold were considered similar levels of expression.

Total RNA extraction from MDA-MB-231 was carried out using the RNeasy Mini Kit (Qiagen). For cDNA synthesis, total RNA (1 μg) was transcribed using random hexamers (Invitrogen, Carlsbad, CA), and SuperScript III reverse transcriptase (Invitrogen) following the manufacturer’s protocol. For quantification of OXPHOS-related genes, the cDNA amplication program included a denature at 95°C for 3 min, followed by 40 cycles of 95°C for 10 s; 58°C for 30 s. For MtDNA detection, total cellular DNA was isolated with DNAeasy Blood and Tissue Kit (Qiagen). Mitochondrial DNA content was determined by qPCR by using comparing the mitochondrially encoded Cox2 gene to an intron of the nuclear-encoded β-globin (HBB) gene. All qPCR was performed using an iQ5 iCycler (Bio-Rad) according to the manufacturer’s instructions. Data were analyzed using Bio-Rad iQ5 Optical System Software v2.0. All products yielded a single band with the predicted size. All primers are listed in Additional file 1: Table S1 and all products yielded a single band with the predicted size.