Dna engine

Quantitative polymerase chain reaction was performed using the DNA Engine with the Chromo 4 detector (MJ Research, Waltham, MA). 1× SuperMix (Platinum SYBR Green quantitative polymerase chain reaction kit, Invitrogen, Carlsbad, CA), complementary DNA, and each primer pair were added to the reaction tube to reach a final volume of 20μl. Target gene expressions were calculated by their ratios to the housekeeping gene, hypoxanthine-guanine phosphoribosyl transferase (HPRT). The sequence of primer sets used for target genes could be found in Table 1.

Quantitative RT-PCR was performed using the DNA Engine with the Chromo 4 detector (MJ Research, Waltham, MA). Target gene expression was calculated relative to the housekeeping gene, hypoxanthine-guanine phosphoribosyl transferase (HPRT). The sequence of primer sets used for target genes were listed in Supplementary Table 2.

The LAMP reaction was performed in a reaction mixture, as described previously14 (link). Briefly, the reaction mixture contained 1 × Bst DNA polymerization buffer, 1 U of Bst DNA polymerase (New England Biolabs, Frankfurt, Germany), 0.5 μM outer primers (F3 and B3 primers each), 4 μM inner primers (FIP and BIP primers each), and 200 μM dNTPs each. Finally, the different amount of sample’s genomic DNA was used to each LAMP reaction. The mixtures were reacted at 60–65 °C for 60 min in a heating block (DNA engine, Biorad, CA, USA).

Pellets in ITS + 10% FBS group and 10% FBS alone group were collected at different time points (0 day, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks; 3 pellets/group/time point) and then minced. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA was reverse transcribed into cDNA.
For semi-quantitative PCR analyses, the cycling reactions were processed in a peltier thermal cycler (DNA Engine^®, Bio-RAD, Hercules, CA, USA) and the RT-PCR products were visualized using Gel Image System (Biospectrun AC, UVP, Upland, CA, USA).
For quantitative real-time PCR, the expression levels of selected genes were analyzed using a LightCycler 480 system with a SYBR green kit (Roche Molecular Biochemical, Mannheim, Germany).
The primer sequences used in this study are shown in Table 2. Primer sequences were quoted from references [27 (link),28 (link)] or designed using Gene Runner software (version 3.05, Hastings Software Inc. Hastings, NY, USA). The specificity of sequences was verified using the basic local alignment search tool (BLAST) of the National Center for Biotechnology Information (NCBI) online database. The expression level of each gene was normalized to GAPDH.

Genomic DNA was extracted from the bacterial cultures using the DNeasy Blood & Tissue Kit (Qiagen Multiplex PCR Kit, Hilden, Germany). Primer sequences were published by the Center for Disease Control and Prevention (CDC) (http://www.cdc.gov/streplab/pcr.html). A total of 28 primers were grouped into nine multiplex reactions. Primers used in this study are listed in Additional file 1: Table S1 and PCR reactions conditions were performed as described previously [15 (link)], with minor modifications. Briefly, multiplex PCR was performed in 50 μl volumes and each reaction mixture contained the following reagents: 5 μl 10 × PCR buffer (Qiagen Multiplex PCR Kit), 5 μl DNA sample, 1 μl dNTPs (50 μM/μl), 1 μl Taq (5 U/μl), 37 μl H₂O; 0.5 μl Primer F; and 0.5 μl Primer R. PCR tubes were placed in a thermocycler (DNA Engine, BioRad, Hercules, California, USA) programmed for initial denaturation at 94 °C × 4 min, followed by 30 amplification cycles at 94 °C × 45 s, annealing at 54 °C × 45 s, extension at 72 °C × 2 min, and a final extension at 72 °C × 10 min. PCR products were electrophoresed in a 2 % agarose gel at 50–100 volts for 40 min, stained with ethidium bromide, and visualized by transillumination (Ultra-Violet Products Ltd., Upland, California, USA).

B-cell clonality (IgH gene rearrangements) was assessed using fragment analysis for V-D-J rearrangements of IgH (FR1, FR2, FR3), as previously described (19 (link)). The reaction mixture included 100–200 ng of DNA. PCR conditions: initial denaturation at 95°C (5 min), 35 cycles of PCR at 92°C (35s), 60°C (35s) and 72°C (35s) and final elongation at 72°C (10 min). PCR was performed on an automatic thermal cycler DNA Engine (BioRad, Hercules, USA). The ABI PRISM 3130 Genetic Analyzer (Applied Biosystems, USA) was used for fragment analysis of PCR products. Results were visualized using the GeneMapper v. 4.0 (Applied Biosystems, USA).