Ambion trizol reagent

For quantitative real time PCR (Q-PCR) analysis, total RNA was isolated from cell or tissue samples using the Ambion Trizol reagent obtained from Life Technologies (Carlsbad, CA, USA) following the manufacturer’s protocol. cDNA was synthesized from 1 ug of total RNA with the high-capacity cDNA reverse transcription kit purchased from Applied Biosystems (Foster City, CA, USA). Q-PCR was performed using SYBR Green PCR master from Applied Systems using the following mouse primers: 18S; 5′-AACCCGTTGAACCCCATT-3′ and 5′-CCATCCAATCGGTAGTAGCG-3′, SF-1; 5′-CCCTTATCCGGCTGAGAATT-3′ and 5′-CCAGGTCCTCGTCGTACGA-3′, StAR; 5′-GTGGCTGCCGAAGACAATC-3′ and 5′-AGGTGGTTGGCGAACTCTATC-3′, Cyp11a1; 5′-TGAATGACCTGGTGCTTCGT-3′ and 5′-GGCAAAGCTAGCCACCTGTA-3′, Cyp11b1; 5′-GAT ACA GAT CCT GAG GGA GC-3′ and 5′-CCG GCA ACG TCA CAA ACA CA-3′, Cyp11b2; 5′-CAGGGCCAAGAAAACCTACA-3′ and 5′-ACGAGCATTTTGAAGCACCT-3′, Hsd3b2;5′- CCAGGGCATCTCTGTTGTCAT-3′ and 5′-GGTTCTGGGTACCTTTCAGATTGA-3′. 18S was used for normalization of gene expression.

RNA for qRT-PCR analysis was prepared from two independent experiments of infected RAW 264.7 cells at 3 and 6 h post-infection at MOI 10. Bacterial RNA was isolated using Ambion TRIzol reagent (Life technologies) and Direct-zol RNA Miniprep kit (Zymo Research). The cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad) following the manufacturer’s protocol (priming: 25°C, 5 min; reverse transcription: 42°C, 30 min; RT inactivation: 85°C, 5 min; and store temperature: 4°C). The concentration and purity of cDNA were measured and normalized to 100 ng/mL for qRT-PCR step. The primers for qRT-PCR indicated in Table 1 were designed and then evaluated for specificity by conventional PCR using Q5 High-fidelity DNA polymerase (New England Biolab). Gene expression was quantified using QuantiNova SYBR green (Qiagen) following the PCR cycling program as follow: initial heat activation step at 95°C for 2 min; two-step 40 cycles of 5 s at 95°C and 30 s at 60°C. The threshold cycle and melting curve of each gene were automatically established and recorded by the software CFX Maestro Software (version 4.0). Relative gene expression level of each gene in ΔbicA mutant was normalized to wild-type strain using the 2^−ΔΔCt method with 16S rRNA as reference gene.

To isolate total RNA a modification of the method described by Wang et al. (2011 (link)) was used. Briefly, samples were first manually ground into crude powder in liquid nitrogen using a mortar and pestle, and then 2 g of crude powder was, respectively, weighed and further ground for 5 min in liquid nitrogen using a Retsch RM100 grinder (Retsch GmbH, Haan, Germany). After grinding, 0.3 g frozen fine powder was weighed into each 50-mL tube and mixed with 3 mL of Ambion TRIzol reagent (Life Technologies, Carlsbad, USA). Subsequent procedures were conducted in accordance with the method of Wang et al. (2011 (link)). After the extraction, an Ambion MEGAclear kit (Life Technologies, Carlsbad, USA) was used to purify the isolated RNA. The RNA concentration and integrity were estimated using an Agilent 2100 bioanalyzer and RNA 6000 Nano kit (Agilent Technologies, Santa Clara, USA). The prokaryotic total RNA nano assay protocol was used, as prokaryotes account for the majority of RNA in rumen contents (Yu and Forster, 2005 ).
Five hundred nanogram of isolated total RNA from each sample was used to synthesize the first-strand cDNA using an Invitrogen SuperScript III RT kit (Life Technologies, Carlsbad, USA), and the cDNA synthesis reactions were stored at −20°C until further analysis was performed.

Total RNA was isolated using Ambion Trizol reagent (Life Technologies, CA, USA) and reverse transcribed to cDNA using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystem, CA, USA) in accordance with the manufacturer's instructions. For reverse transcription PCR (RT-PCR), cDNAs were amplified using TaKaRa ExTaq (TaKaRa Bio Inc., Shiga, Japan) and the PCR products were analysed on 1.5% agarose gel. For quantitative real-time PCR, cDNA and primers were prepared with a Power SYBR Green PCR Master Mix (Applied Biosystem, Warrington, UK) according to the manufacturer's instructions.
For mitochondrial DNA content analysis, total DNA was extracted using DNAzol Reagent (Invitrogen, CA, USA) according to the manufacturer's instructions. Mitochondrial DNA was amplified using primers specific for the mitochondrial cytochrome c oxidase subunit 2 (COX2) gene and normalized to genomic DNA by amplification of the ribosomal protein s18 (rps18) nuclear gene65 (link).
The primer sequences used for reverse transcription–PCR and quantitative real-time PCR can be found in the Supplementary Table 1.

Frozen whole CNS tissue was shipped on dry ice to University of Vermont Cancer Center DNA Analysis Facility (Burlington, VT) for extraction. Tissue was stored at −80°C until RNA extraction. Frozen tissue was homogenized in 1 mL of Ambion® TRIzol® Reagent (LifeTech, #15596-026) using a sterile, autoclaved mortar, and pestle. RNA was extracted per the Ambion® TRIzol® protocol (LifeTech, MAN0001271) and purified using Qiagen RNeasy Mini Kit (Qiagen #74104) per manufacturers protocol (Qiagen RNeasy Mini Handbook, 06/2012).

Basolateral supernatants from A2EN cells that had been exposed to cell-free or cell-associated HIV were centrifuged at 20,000×g for 1 h at 4°C. The RNA was then purified using Ambion Trizol^® Reagent (ThermoFisher, Waltham, MA) in accordance with the manufacturer's instructions. RNA templates were used as standards. The entire sample of RNA from each condition was reverse-transcribed, and polymerase chain reaction (PCR) amplification of the HIV-1 pol gene was performed on each sample, as previously described [47 (link)]. Two HIV-negative samples, one HIV-positive sample, and negative experimental controls were included in each assay. HIV levels are reported as copies per replicative sample.

We conducted brain collection and RNA extraction according to a previous study (18 (link)). After the end of the social interaction test (10–11 weeks of age), whole brains were collected. We examined the whole brain in the present study because the precise brain regions that are associated with ASD have not yet been clearly defined. Total RNA was extracted from the whole brain and homogenized in Ambion TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) using a homogenizer. RNA was isolated using chloroform and precipitated using isopropyl alcohol. The quality of RNA was assessed with Nanodrop 1000 (Thermo Fisher Scientific). All of the RNA samples had an A_260/280 ratio between 2.01 and 2.02 and A_230/260 ratio between 2.26 and 2.31.