Rotor gene rg 3000

RNA was isolated using Tri (Sigma) according to the manufacturer’s recommendations. One microgram of total RNA was treated with DNAseI (New England Biolabs Inc.) and reverse transcribed using random hexamer primers (Fermentas). One microlitre of the resulting cDNA was used for real-time PCR using Rotor-Gene SYBR Green PCR kit on a Rotor-Gene RG-3000 (Qiagen). Gene transcripts were normalised to the Gapdh RNA content (primers listed in Additional file 8). All results are based on the ΔΔCT method and represent the mean of at least three independent experiments.

Overnight cell cultures of ZJ-T, Δhfq-T and hfq⁺-T were diluted to OD₆₀₀ = 0.01 in TSB medium and grown to early exponential phase (OD₆₀₀ = 0.5) and bacterial cells were collected. All reagents were from Takara Bio Inc. Total RNA was isolated by using RNAiso Plus, DNase-treated and 1 μg of RNA was reverse-transcribed with PrimeScript^TM RT reagent Kit. PCR amplification was then carried out on a RotorGene RG-3000 (Qiagen) using SYBR Premix Ex Taq™ with primers specific to the genes to be assayed (S1 Table). Three housekeeping genes (recA, uvrA and gyrA) were used as internal controls. Relative gene expression was calculated by the 2^−ΔΔCt method [33 (link)] and normalized to the wild type ZJ-T value. Measurements were done in triplicates. Statistical significance was assayed by one-way ANOVA LDS method (*p < 0.05, **p < 0.01, ***p < 0.001).

RNA was isolated using Sepasol-RNA I Super G (Nacalai Tesque, Kyoto, Japan) or the CellAmp Direct RNA Prep Kit (Takara Bio Inc., Ootsu, Japan), and was subsequently reverse-transcribed using the PrimeScript II First Strand cDNA Synthesis Kit (Takara Bio Inc.) [22 (link),23 (link)]. RT-PCR using QuantiFast SYBR Green PCR (Qiagen, Venlo, The Netherlands) was performed on a Rotor Gene RG-3000 (Qiagen). The relative mRNA expression levels were determined by the comparative C_t method; expression levels of individual genes were normalized against the levels of the reference gene HPRT, which encodes hypoxanthine guanine phosphoribosyl transferase. The following primer sets and annealing temperatures were used: survivin, 5- CCAGTGTTTCTTCTGCTTCAA-3 and 5-GAATGCTTTTTATGTTCCTCTATG-3 at 60°C; HPRT, 5- TGACCTTGATTTATTTTGCATACC-3 and 5-CTCGAGCAAGACGTTCAGTC-3 at 60°C [11 (link),12 (link)].

The levels of each mRNA were analyzed by real-time PCR. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed into cDNA by a TOPscript RT DryMIX kit (Enzynomics, Seoul, Korea). Equal amounts of cDNA were amplified in a 10 µl reaction solution containing 1  × SYBR PCR master mix (Takara Bio Inc., Otsu, Japan) and 10 µM primers. Real-time PCR was carried out by a Rotor Gene RG-3000 (Corbett Life Science, Sydney, Australia) according to the PCR conditions as follows: initial denaturation for 5 min at 95 °C, followed by 35 cycles of 20 s at 95 °C, 50 s at 58 °C, and 40 s at 72 °C. The primers were tumor necrosis factor (TNF)- α, 5′-TCCCAAATGGCCTCCCTCTCA-3′ and 5′-GTGGTTTGCTACGACGTGG-3′; interleukin (IL)-1 β, 5′-AAACCTTTGACCTGGGCTGTCC-3′ and 5′-TGCCTGCCTGAAGCTCTT GTT-3′; monocyte chemotactic protein (MCP)-1, 5′-TGCAGTTAACGCCCCACTCAC-3′ and 5′-CAGCTTCTTTGGGACACCTGC-3′; GAPDH, 5′-CATGGCCTTCCGTGTTCCTA-3′ and 5′-CCTGCTTCACCACCTTCTTGAT-3′.

pfmdr1 gene copy numbers were determined through real-time PCR using fluorescent TaqMan probe-based gene expression. Primer pairs of pfmdr1 [15 (link)] and the housekeeping gene seryl-t-rna-synthetase were designed to match in length and nucleotide content of the two amplified gene regions. In addition, TaqMan probes with a FAM or VIC dye on the 5′ end and a TAMRA quencher on the 3′ end were added to the reaction mix (Applied Biosystems, USA). The primers used for quantifying copy number were: PF-F: 5′-TTAAGTTTTACTCTAAAAGAAGGGAAAACATA, PF-R: 5′-TCTCCTTCGGTTGGATCATAAAG, PF-FAM: 5′-FAM-CATTTGTGGGAGAATCAGGTTGTGGGAAAT-TAMRA, seryl_F:5′-GATTTATTAAGAAAAATAGGTGGAGCTA, seryl_R: 5′-TATAGCATTATGTAATAAGAAACCTGC, seryl_probe: 5′ VIC-AAGGTATACAAGTAGCAGGTCATCGTGGTT-TAMRA. The reaction mix contained 20 ng DNA, 300 nM primers, 250 nM TaqMan probes, 300 μM dNTPs, 2 mM MgCl₂ and 2.5 units/reaction HotStarTaq DNA Polymerase (Qiagen). Samples were prepared in triplicates. For the reaction, the initial activation step was at 94°C for 3 min., followed by 40 cycles of 94°C for 1 min., 60°C for 1 min. and 72°C for 30 sec. Fluorescence was recorded after each elongation step. Real-time PCR was carried out in a Rotor-Gene RG-3000 (Corbett Research, Toronto, ON, Canada). Copy numbers were determined relative to 3D7, which is known to have only one pfmdr1 gene copy [16 (link),17 (link)].

mtDNA copy number was determined as described, by calculating the ratio of mtDNA to nuclear DNA (ACTB) per cell in relation to standard curves of known concentrations, ranging from 10⁻¹ ng/μL to 10⁻⁸ ng/μL [42 (link)]. Quantitative PCR was performed on a Rotor Gene RG-3000 (Corbett Research, Mortlake, Australia), with RotorGene software (v6.0) used for data acquisition and analysis. Standard curves were used with a correlation coefficient of R² > 0.9 and an efficiency coefficient of R > 0.95. Mitochondrial DNA copy number per cell was calculated as follows:

Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA, Cat#15596-018) and reverse-transcribed into cDNA using a TOPscript RT DryMIX Kit (Enzynomics, Seoul, Korea, Cat#RT201) to evaluate the levels of mRNA as described previously [24 (link)]. Briefly, real-time PCR was performed using equal amounts of cDNA in a Rotor Gene RG-3000 (Corbett Life Science, Sydney, Australia). The reaction was carried out in a volume of 20 μL and contained 10 pM primers and 2× Real-Time PCR Smart Mix (Solgent, Daejeon, Korea, Cat#SRH81-M40h). Following an initial denaturation for 10 min at 95 °C, the reactions were amplified by 45 cycles of 25 s at 95 °C, 45 s at 57 °C, and 35 s at 72 °C. The following primers were used: SIRT1 (NM_012238.5), 5′-TAGCCTTGTCAGATAAGGAAGGA-3′ and 5′-CTCGTACAGCTTCACAGTCAAC-3′; and GAPDH (NM_002046.7), 5′-CCTGCTTCACCACCTTCTTGAT-3′ and 5′-CATGGCCTTCCGTGTTCCTA-3′. The level of SIRT1 mRNA in each sample was normalized against that of GAPDH mRNA. The fold change in target gene expression relative to GAPDH expression was determined as described previously [25 (link)].