Goscript reverse transcription kit

RNA was extracted from incubation slurries as described previously (46 (link), 61 (link)). Isopycnic centrifugation and gradient fractionation were conducted to separate ¹³C-labeled from unlabeled RNA. In detail, 500 to 1000 ng of RNA was loaded with formamide (240 μl), cesium trifluoroacetate solution (6 ml), and gradient buffer solution, followed by ultracentrifugation using an Optima L-90 XP ultracentrifuge (Beckman Coulter, Brea, CA, USA). After centrifugation at 124,000g at 20°C for 65 hours, 14 fractions (~410 μl for each fraction) were collected from each sample. At the same time, mixed fully ¹³C-labeled and unlabeled RNA from Escherichia coli was used as standard during density separation for defining heavy and light fractions. cDNA was obtained by reverse transcription of RNA using GoScript reverse transcription kit (Promega, Madison, Wisconsin, USA). A combination of cDNA from fractions 4 and 5 (heavy), 6 and 7 (middle), 8 and 9 (light), and 10 and 11 (ultralight) was performed for 16S rRNA gene sequencing, respectively.

Waddlia-infected cells were harvested in TRIzol (AmbionR, Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) at different times post infection, as previously described (56 (link)). RNA-extraction was performed according to the manufacturer’s instructions. RNA was re-suspended in 60 µL of water and treated with the DNA-free DNA removal kit (Invitrogen, Thermo Fisher Scientific) to remove DNA contaminations. Random primers and the GoScript Reverse Transcription kit (Promega, Duebendorf, Switzerland) were used to obtain cDNA. Gene expression was analyzed by quantitative PCR on 4 µL of fivefold diluted cDNA using iTaq SYBR Green (Bio-Rad, Cressier, Switzerland), and the primers listed in Table 3. Reactions were conducted on a QuantStudio3 system (Applied Biosystems, Thermo Fisher Scientific) using the following cycling conditions: 10 min at 95°C, 40 cycles of 15 s at 95°C, and 1 min at 60°C. Results were analyzed (with the ΔΔCt method) using 16S rRNA as endogenous control at 32 hpi (in the case of kinetic experiments) or the untreated sample (in the case of aberrant bodies) as reference sample. For fold changes ≤0.5 or ≥2, significance was assessed by paired t-test on ΔCt values.

For gene expression analysis the total RNA extraction was performed by using Trizol^® (ThermoFisher Scientific, Beverly, MA, USA) following the manufacturer protocol. After extraction, total RNA concentration was determined by using a NanoDropTM Lite spectrophotometer (ThermoFisher Scientific, Beverly, MA, USA). The integrity of total RNA was checked by using 1% agarose gel. After quality check, total RNA was reverse-transcribed into cDNA by using a Go Script Reverse Transcription kit (Promega, Madison, WI, USA) according to the manufacturer protocol.
Primers used for RT-qPCR of the target and housekeeping genes are described in Table 3. RT-qPCR reactions were performed in QuantStudio^® 3 (Applied Biosystems, Thermo Fisher Scientific).
The threshold cycle (Ct) values obtained were normalized (ΔCt) based on the Ct value of glyceraldehyde-3-phosphate dehydrogenase. Gene of interest relative expression was calculated by 2^−ΔΔCt [24 ].

Total RNA of the hippocampus was isolated using a Eastep® Super RNA extraction kit (Promega, Shanghai, China) followed by reverse transcription with a GoScript™ Reverse Transcription Kit (Promega, Shanghai, China) according to the manufacturer’s protocol. Finally, RT-PCR was performed with GoTaq® qPCR Master Mix (Promega, Shanghai, China) and specific primers (Sangon Biotech, Shanghai, China). The relative fold change in gene expression was calculated with the 2^−ΔΔCt method with GAPDH as the internal control [34 (link)]. The primers used to detect TNF, IL-6, IL-1β, KCC2, and GAPDH mRNA were as follows:

TNF forward: 5’-CTGTGAAGGGAATGGGTGTT-3’;

TNF reverse: 5’-CAGGGAAGAATCTGGAAAGGTC-3’;

IL-6 forward: 5’-GGCCCTTGCTTTCTCTTCG-3’;

IL-6 reverse: 5’-ATAATAAAGTTTTGATTATGT-3’;

IL-1β forward: 5’-AGTTGACGGACCCAAAAG-3’;

IL-1β reverse: 5’-AGCTGGATGCTCTCATCAGG-3’;

KCC2 forward: 5’- AGGTGGAAGTCGTGGAGATG-3’;

KCC2 reverse: 5’-CGAGTGTTGGCTGGATTCTT-3’;

GAPDH forward: 5’-GACATGCCGCCTGGAGAAAC-3’;

GAPDH reverse: 5’-AGCCCAGGATGCCCTTTAGT-3’.

mRNA was extracted from cultured MDM using RNAqueous Micro-kit (Ambion) according to the manufacturer’s instructions and treated with DNase (Ambion) and DNase inhibitor (Ambion). Single strand cDNA was synthesised using the GoScript Reverse Transcription Kit (Promega) according to the manufacturer’s recommendations. iTaq Universal SYBR® Green Supermix (Bio-Rad) and forward and reverse primers (Sigma) (final concentration of each 0.5pM) were prepared in triplicate in 96 well plates prior to addition of cDNA diluted 1 in 10. Q-PCR performed using LightCycler 480 (Roche). Primer sequences and efficiencies are detailed in Table 2.Table 2

Primer details [47 (link)]

Gene	Forward Sequence	Reverse Sequence	No. bp	Efficiency
nos2	TTGAGATCAACGTCGCTGTG	CATGATGGTCACGTTCTGCT	56	98
arg1	ATGTGGACCCTGGGGAAC	ATGTTTCTTCCATCACCTTGC	105	101
gapdh	CACCATCTTCCAGGAGCGAG	CCAGCATCACCCCACTTGAT	51	100

The measured change in transcription levels of nos2 and arginase-1 following stimulation was compared with that of the reference gene, glyceraldehyde 3-phosphate dehydrogenase (gapdh) and fold changes were calculated using the delta-delta C_t method where the ratio R is determined by the equation below [46 ]. $$\mathrm{R}=2^{‐\left\{\mathrm{\Delta Cp}\text{sample}-\mathrm{\Delta Cp}\text{reference}\right]}$$