Dna thermal cycler

PFCs were stimulated as described above for 12 h. Total RNA was isolated using an RNeasy mini kit (Qiagen, Valencia, CA), and residual DNA was removed using RNase-free DNAse (Qiagen). Reverse transcription of total RNA to cDNA was performed at 37°C using a Reaction Ready-First Strand cDNA Synthesis kit (Promega). Amplification of cDNA was conducted in a DNA thermal cycler (Biometra, Germany) using the following conditions: 95°C for 45 s, 55°C for 45 s, and 72°C for 45 s for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 95°C for 45 s, 51°C for 45 s, and 72°C for 45 s for IL-21. PCR was repeated for 35 cycles for both GAPDH and IL-21. Primer sequences were as follows: IL-21 sense 5’- GAG-TGG-TCA-GCT-TTT-TCC-TGT-T-3’, IL-21 anti-sense 5’-AGG-AAT-TCT-TTG-GGT-GGT-TTT-T-3’, GAPDH sense 5’-GCA-TGG-CCT-TCC-GTG-TCC-3’, and GAPDH anti-sense 5’-TGA-GTG-TGG-CAG-GGA-CTC-3’.

Three sense TCR Vγ primers and a single Cγ reverse primer, or eight TCR Vδ sense primers and a single Cδ primer were used in an unlabeled PCR to amplify the TCR Vγ and Vδ subfamilies, respectively. PCR was performed as described in our previous report [38 (link)]. Aliquots of the cDNA (1 μl) were amplified in 20 μl with 1 of the 3 Vγ primers with a Cγ primer or 1 of the 8 Vδ primers with a Cδ primer. The final mixture contained 0.5 μM sense and antisense primer, 0.1 mM dNTP, 1.5 mM MgCl₂, 1× PCR buffer and 1.25 U Taq polymerase (Promega, USA). The amplification was performed in a DNA thermal cycler (BioMetra, Germany) with a 3 min denaturation at 94°C and 40 PCR cycles. Each cycle consisted of 94°C for 1 min, 60°C for 1 min and 72°C for 1 min with a final 7 min elongation at 72°C. All PCR products were analyzed in a 1.5% agarose gel stained with ethidium bromide and then stored at 4°C prior to GeneScan analysis.

For detection of β-lactamase genes responsible for imipenem-resistance, rapid genomic DNA was prepared from about five colonies heated in 100 mL distilled water (95°C for 10 min) followed by a centrifugation step of cell suspension at 12.000 rpm for 5 min; then supernatant was taken as a source of template DNA. PCR amplification was carried out by using DNA thermal cycler (Biometra, Singapore) using a specific primer for bla_VIM, bla_TEM, bla_SHV, bla_CTX-M-1, bla_CTX-M-9, and bla_CTX-M-8/25 (Table 1), in a 50 μL volume containing 10x PCR buffer, 2 mM deoxynucleoside triphosphates, 3.4 pmol of each primer, 2.5 mM MgCl₂, 1 U Taq DNA polymerase, and 1 μL of genomic DNA [12 (link)]. Amplification was carried out as follows: initial denaturation at 94°C for 10 minutes, followed by 40 cycles of DNA denaturation at 94°C for 40 seconds, primer annealing at 60°C for 40 seconds and primer extension at 72°C for 1 minute, and a final elongation step at 72°C for 7 minutes. The annealing temperature was optimal at 55°C instead of 60°C for amplification of bla_VIM. Amplicons were then visualized after running in 2% agarose gel at 100 V for 30 mins. A 50–1000 bp DNA ladder (USA) was used as a size marker. Finally, PCR products were purified with innuPREP PCRpure kit (Analytik Jena, Germany) and subjected to direct sequencing via GATC Company by use of ABI 3730xl DNA sequencer.

For the gene expression of chemokine CCL20 in uterus and blood, uterus and blood were collected as described above. Total RNA were extracted by Trizol reagent (Invitrogen) and then reverse-transcribed with a cDNA Synthesis Supermix (novoprotein scientific Inc) according to the manufacturer’s instruction. Amplification of cDNA was conducted in a DNA thermal cycler (Biometra, Germany). The following sense and antisense primers for each molecule were used: CCL20 Forward: 5′-CTCCTGGAGCTGAGAAT-3′, reverse: 5′-C ATCTTCTTGACTCTTAGGC-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward: 5′-TCAATGAAG GGGTCGTTGAT-3′, reverse: 5′-CGTCCCGTAGACAAAATGG T-3′. The ratio of CCL20 over GAPDH was calculated according to the relative intensities of the bands revealed under UV illumination with Bio-1D software (VilberLourmat, Marne la Vallee, France).

Tissues were homogenized individually and dissolved in Trizol (Invitrogen, Carlsbad, CA, USA) to extract RNA. BALF was centrifuged at 1800 rpm and the supernatants were harvested and centrifuged at 4000 rpm; the methanolysis method was used to isolate RNA. The concentration of RNA was measured by NanoDrop 2000 spectrophotometer (Thermo Fisher, USA) and reverse transcribed with a RT Reagent Kit (Novoprotein, China). Amplification of cDNA was conducted in a DNA thermal cycler (Biometra, Germany). SYBR qPCR Supermix Plus (Novoprotein, China), primers, and cDNA were added in 8 straight tubes in StepOnePlus instruments (Thermo Fisher) to get the corresponding results with StepOnePlus™ Software v2.3. Primer sequences are listed in Table 2.

DNA samples were isolated from peripheral blood using the QIAamp DNA blood kit (QIAGEN GmbH, Hilden, Germany). Genotyping of the FTO SNPs (rs9939609, rs8050136 and rs1421085) was performed using direct sequencing. The PCR-RFLP assay was used to determine the genotype of SNP rs9939506. PCR was carried out in a DNA thermal cycler (Biometra, Goettingen, Germany) using the primers: forward: 5'-CAAAGGTGGGCATAGAGATTG-3'; reverse: 5'-AAGGATTTCTGAGGGACACA-3'. PCR was performed after the first denaturation at 95°C for 2 min; each cycle consisted of denaturation at 95°C for 20 sec, annealing at 62°C for 20 sec, and extension at 72°C for 30 sec. The number of total PCR cycles was 30. To assess genotyping reproducibility, randomly selected 20% DNA samples were re-genotyped with 100% concordance.

Microglia were collected from the seizure and control groups of mice at different time points after stimulation with or without LPS (1 μg/ml). Total RNA was extracted and the expression of IL-10 and IL-1β was detected by RT-PCR. Briefly, total RNA was purified with an RNAeasy mini kit (Tiangen, Beijing, China), and assessed for quality and quantity on a spectrophotometer (Beckman Coulter, Brea, CA, USA). Reverse transcription was carried out in 20 μl reaction volume with a High Capacity Reverse Transcription Kit (Applied Biosystems, Foster City, CA) following the manufacturer’s instructions. PCR was carried out on a DNA thermal cycler (Biometra, Germany) at 95 °C (5 min), 95 °C (45 s), 58 °C (45 s), and 72 °C (45 s), followed by 20–25 cycles of 72 °C for 5 min. GAPDH was used for normalization. The primers (TAKARA, Dalian, China) include GAPDH, 5′-AAATGGTGAAGGTCGGTGTGAAC-3′ (forward) and 5′-CAACAATCTCCACTTTGC CACTG-3′ (reverse); IL-10, 5′-GCCAGAGCCACATGCTCCTA-3′ (forward) and 5′-GATAAGG CTTGGCAACCCAAGTAA-3′ (reverse); IL-1β, 5′-TCCAGGATGAGGACATGAGCAC-3′ (forward) and 5′-GAACGTCACACACCAGCAGGTTA-3′ (reverse). PCR products were assessed by 1.5% agarose gel electrophoresis, and visualized with ethidium bromide under UV light.