T3 thermocycler

Total RNA was extracted from the samples using TRIzol (Invitrogen, USA) reagent according to the manufacturer’s instructions. RNA concentration was determined by measuring the optical absorbance of the samples at 260 nm. The extracted RNA sample (2 μg) was initially reverse transcribed for first strand cDNA synthesis using a PrimeScript 1st Strand cDNA synthesis kit (TaKaRa, China). The reactions were performed in a T3 thermocycler (Biometra, Germany). Real-time PCR was performed using a quantitative real-time amplification system (ABI PRISM 7500 Fast Real-time PCR System; Applied Biosystems, Foster City, CA, USA). SYBR Premix Ex Taq (Tli RNaseH Plus) (Takara, China) was used in each reaction. Adipogenic differentiation markers, namely, adipocyte Protein 2 (ap2), CCAAT/enhancer-binding protein alpha (C/EBP α), lipoprotein lipase (LPL), and peroxisome-proliferating activated receptor γ (PPAR γ), were evaluated (see Table 1). The relative expression level of each gene of interest was calculated by normalizing the quantified cDNA transcript level (cycle threshold) to that of GAPDH using the ABI PRISM 7500 Fast Real-time PCR System.

Genotyping was done in order to differentiate a recrudescence (same parasite strain) from a newly acquired infection (different parasite strain). The P. falciparum merozoite surface protein 1 (Pfmsp-1), P. falciparum merozoite surface protein 2 (Pfmsp-2) and P. falciparum glutamate-rich protein (Pfglurp) genes were used to discriminate reinfections from recrudescence as previously described [40 , 41 (link)]. Summarily, DNA fragments obtained from amplification of baseline samples (day 0) and on the day of recurrent parasitemia were compared according to band size and number, considering the 3 families of Pfmsp-1 (MAD20, RO33, K1), the 2 families of Pfmsp-2 (FC27, 3D7/IC) and single family type of Pfglurp. Cases were categorized as new infections when there were no common bands between day 0 and the day of recurrent parasitemia. However, when there was at least one common band between baseline sample and that of the day of parasite reappearance for any of the 3 markers (even if there were additional bands on day 0) the case was recrudescence. Malaria parasite infected cases were considered not to be clinical failures if their recurrent parasitemia were classified as new infections rather than recrudescent infections. The gene amplification was done using the Biometra T3 Thermocycler (United Kingdom).

Genomic DNA was extracted and purified from peripheral blood with DNA Extractor WB Kit (Wako Pure Chemical Industries, Ltd. Japan) according to the product description. rs2431697 polymorphism was determined based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. A 260-bp DNA fragment around rs2431697 was amplified with the primer pair: sense primer: 5′-AGAGGGGGTGAAAGAAGGAA-3′ and antisense primer: 5′-TTCTCAGTGCCAATGTGAGG-3′. The reaction mixtures contained 10 ng genomic DNA, 10 pmol of each primer, and double-diluted Taq 2× Master Mix (New England Biolabs, UK) in a total 20-μl volume. The reactions were carried out in a T3 thermocycler (Biometra, Göttingen, Germany). The reaction conditions were as follows: denaturation at 94 °C for 5 min, followed by 35ccycles of denaturation at 94 °C for 30 s, annealing for 1 min at 57 °C and extension at 72 °C for 45 s, and a final extension at 72 °C for 5 min. PCR products were subsequently digested by Taq I (New England Biolabs, UK) at 65 °C for 2 h and separated on a 3 % agarose gel. The rs2431697 T allele yielded 61- and 199-bp fragments, and C allele yielded a single 260-bp fragment.

RNA was isolated from PBMNC or CD3-enriched, CD3⁺/CD8⁺ and CD3⁺/CD4⁺ T-cells transcribed into cDNA. TCR-repertoire analyses were done using 25 vß- family specific primers, including controls for the constant region of the TCR and human ß-actin, as described (Naumov et al., 1995 (link)). RT-PCR for detection of the bcr-abl fusion transcript was performed as proposed by the BIOMED-1 nested PCR on Taqman concerted action (Van Dongen et al., 1999 (link); Borchers et al., 2011 (link)). PCR was performed with the T3 thermocycler (Biometra). Donor chimerism was analyzed by PCR amplification of highly polymorphic short tandem repeat (PCR-STR) sequences in peripheral blood and/or bone marrow samples as described earlier (Briones and Amils, 1998 (link)).

Cloning, restriction enzyme analysis, and transformation of E. coli were performed using established procedures [14 ]. To amplify the AHL synthase gene (halI) and the AHL receptor gene halR by PCR, the reaction consisted of 25 mM MgCl₂, 5.0 μL of 10X buffer Ex Taq, 25 mM deoxynucleotide triphosphates (dNTPs), 25 μM of each primer, 1 U Ex Taq DNA polymerase, and 40 ng of DNA from H. alvei 068 in a final volume of 50 μL. Primers based on the sequences of halI and halR genes (GenBank accession number AF503776) of H. alvei were constructed (see Table 2) and synthesized by Microsynth (Zürich, Switzerland). PCR reactions were carried out in a T3 thermocycler (Biometra®, Biolabo Scientific Instruments, Zürich, Switzerland).
The M13 Universal and Reverse Primers were used to sequence halI and halR genes cloned into pCR2.1-TOPO.

For the amplification of HEF PCR products of the IDV from 2015 for Sanger sequencing, previously described primers were used [14 (link)], along with the SuperScript^TM III One-Step RT-PCR System with Platinum^TM Taq High Fidelity DNA Polymerase kit (Invitrogen^TM, Thermo Fischer Scientific, Roskilde, Denmark). The RT-PCR was performed in a final volume of 40 µL with 5 µL RNA. A total of 20 µL reaction mix (2X) was mixed with 0.20 µL of each primer (100 µM), 0.80 µL enzyme mix, and 13.7 µL nuclease-free water. The RT-PCR was run on a T3 Thermocycler (Biometra, Fredensborg, Denmark) with the following thermal cycling conditions: 50 °C, 30 min; 94 °C, 2 min; 5 cycles x (94 °C, 10 s; 60 °C, 30 s; 68 °C, 45 s); 35 cycles x (94 °C, 10 s; 57 °C, 30 s; 68 °C, 45 s); 5 cycles x (94 °C, 10 s; 54 °C, 30 s; 68 °C, 45 s); 68 °C, 5 min. The PCR products were visualized on 0.8% E-gel^TM general purpose agarose gels (Invitrogen^TM). The PCR products were subsequently purified with the High Pure PCR Product Purification Kit (Roche, Hørsholm, Denmark). The purified products were then Sanger sequenced with the aforementioned PCR primers at LGC Genomics (Berlin, Germany).