Lightcycler 480 software version 1

Total RNA was isolated from cell cultures using the TRI reagent (MRC, Inc., Cincinnati, OH), and 2 µg of RNA was reverse-transcribed into cDNA using random hexamer primers (Bioneer, Daejeon, Korea) and Superscript III reverse transcriptase (Invitrogen, Grand Island, NY) in a thermal cycler (Eppendorf, Happauge, NY) according to the manufacturer's instructions. Quantitative RT-PCR was carried out in a total volume of 10 µL containing 5 µL of LightCycler® 480 SYBR Green I Master (Roche Diagnostics Ltd., Rotkreuz, Switzerland), 0.5 µM of each primer and 2.5 µL of 1∶10 diluted cDNA using LightCycler® 480 instrument (Roche Diagnostics Ltd). The cycling conditions were: 95°C for 5 min, followed by 45 cycles of 95°C for 10 s, 60°C for 10 s, and 72°C for 10 s. All the samples were carried out in triplicates. The expression levels of each mRNA expression were normalized to the housekeeping gene GAPDH using LightCycler® 480 Software, Version 1.5 (Roche Diagnostics Ltd). Primer sequences for FOXG1, DLX2, NKX2.1, GAD1, calbindin2 (CALB2) and somatostatin (SST) have been previously described [26] (link). Other primer sequences were retrieved from PrimerBank Database http://pga.mgh.harvard.edu/primerbank/[27] (link), with the exception of SLC32A1 which was designed using ProbeFinder software Version 2.49 from Roche Applied Science. Sequences of primer sets were listed in Table S1.

Total RNA from cells and livers was extracted using RNeasy mini kit (Qiagen). One microgram of RNA was retro-transcribed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The qPCR reactions were set up using SYBR Green Master Mix and run in duplicate on a Light Cycler 480 System (Roche). The primer sequences for mouse Tnf were: forward, 5′-ctgaacttcggggtgatcgg-3′; and reverse, 5′-ggcttgtcactcgaattttgaga-3′. The primer sequences for mouse Il6 were: forward, 5′-ccggagaggagacttcacag-3′; and reverse, 5′-cagaattgccattgcacaac-3′. The primer sequences for mouse B2m were: forward, 5′-tggtgcttgtctcactgacc-3′; and reverse, 5′-gtatgttcggcttcccattc-3′. Running program was as follows: pre-heating, 5 min at 95 °C; 40 cycles of 15 s at 95 °C, 15 s at 60 °C, and 25 s at 72 °C. B2m was used as housekeeping gene. Data were analyzed by LightCycler 480 software version 1.5 (Roche).

To detect S. agalactiae in vaginal DNA extracts, a S. agalactiae specific qPCR was carried out, using primers previously described [23 (link)]. The qPCR reactions for S. agalactiae were performed in a final volume of 10 μl, containing 5 μl of LightCycler 480® SYBR Green I Master (Roche), 0.5 μM of both forward primer Sip1 (5’-ATCCTGAGACAACACTGACA-3’) and reverse primer Sip2 (5’-TTGCTGGTGTTTCTATTTTCA-3’), 0.3 μM of probe (5’- 6-FAM–ATCAGAAGAGTCATACTGCCACTTC–TAMRA-3’) (Eurogentec, Liège, Belgium) and 2 μl of DNA extract or 2 μl of HPLC water (as negative template control). Cycling conditions were as follows: 95°C for 5 min; 40 cycles of 95°C for 10 s, 58°C for 15 s and 72°C for 20 s. For the standard series, DNA concentration of the extract of S. agalactiae LMG 14694^T was determined using the Qubit® Fluorometer (Invitrogen, Auckland, New Zealand) and the genomic concentration was calculated based on the GC% content and genome size of the type strain. A tenfold dilution standard series of S. agalactiae LMG 14694^T DNA was prepared by dilution of the DNA stock in HPLC grade water. All standard tenfold dilution series and samples were run in duplicate. Amplification, detection and quantification were carried out using the LightCycler480® platform and the LightCycler® 480 Software Version 1.5 (Roche).

Total RNA was extracted with High Pure RNA Isolation Kit (Roche), according to the manufacturer’s instructions. RNA was quantified in a NanoDrop 2000c (Thermo Scientific) and used for cDNA synthesis. Reverse transcription was performed with High Fidelity cDNA Synthesis Kit (Roche), using Anchored-oligo(dT)18 Primer (Roche) or with the Super Script III First Strand synthesis system (Invitrogen), using random hexamers (Invitrogen). qPCRs were performed in triplicates using LightCycler 480 SYBR Green I Master Kit (Roche) with the following primers: βIII-tubulin (TUBB3; fwd 5′-GGGCCTTTGGACATCTCTTC-3′ and rev 5′-CCTCCGTGTAGTGACCCTTG-3′), glial fibrillary acidic protein (GFAP; fwd 5′-AGAGAGGTCAAGCCCAGGAG-3′ and rev 5′-GGTCACCCACAACCCCTACT-3′) and ribosomal protein L22 (RPL22; fwd 5′-CACGAAGGAGGAGTGACTGG-3′ and rev 5′-TGTGGCACACCACTGACATT-3′). The reactions were performed with LightCycler 480 Instrument II 96-well block (Roche). Quantification cycle values (C_q’s) and melting curves were determined using LightCycler 480 Software version 1.5 (Roche). All data were analyzed using the 2^−ΔΔCt method for relative gene expression analysis47 (link). Changes in gene expression were normalized using the housekeeping gene RPL22 as internal control.

Total RNA was extracted by PerfectPure® RNA Cultured Cell Kit (5 PRIME, Gaithersburg, MD) and reverse transcribed using the Superscript® III First-Strand synthesis system (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions. Primers were designed based on NCBI database. The primer sequences and the individual PCR product sizes are demonstrated in Table 1. Targets were amplified for 40 cycles with denaturation at 95 °C for 15 s and annealing at 60 °C for 15 s and extension at 72 °C for 45 s. For MyD88, cDNA was amplified for 60 cycles of denaturation at 95 °C for 15 s and annealed at 60 °C for 15 s using Lightcycler® 480 software version 1.5 (Roche Applied Science, Mannheim, Germany). Gene expressions were normalized with β-actin (ACTB) and determined by the ∆C_T method.Table 1

Sequences of primers for real-time PCR

Gene	Accession no.	Primer (5′-- > 3′)		Size (bp)
ACTB	NM_001101.3	F	CACACTGTGCCCATCTACGA	162
		R	CTCCTTAATGTCACG CACGA
TLR4	NM_138554	F	TCACAGAAGCAGTGAGGATGAT	140
		R	AAGTAATATTAGGAACCACCTCCA
MyD88	NM_002468	F	TGCAGAGCAAGGAATGTGAC	153
		R	GGTTGGTGTAGTCGCAGACA
IL-6	NM_000600	F	CGGGAACGAAAGAGAAGCTCTA	68
		R	GGCGCTTGTGGAGAAGGAG
IL-8	NM_000584	F	GCCAACACAGAAATTATTGTAAAGCTT	112
		R	AATTCTCAGCCCTCTTCAAAAACTT
XIAP	NM_001167	F	CCATGGCAGATTATGAAGCA	176
		R	TTGTTCCCAAGGGTCTTCAC