Trizol (Ambion Inc., Austin, TX, USA) reagent solution was added to the skin tissue, and total RNA was isolated. The amount and purity of total RNA were determined using 260 nm and 280 nm in spectrometers (Perkin Elmer). According to the manufacturer’s manual, total RNA (1 µg) was synthesized into cDNA using the Superscript™ III Reverse Transcriptase Kit (Bio-Rad, Richmond, CA, USA). The synthesized cDNA was mixed with SYBR Green supermix (Bio-Rad, Richmond, CA, USA). Its amplification was performed using the CFX Connect™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Relative mRNA expression of TNF-α, interleukin (IL)-4, and IL-13 in the skin was assessed using the method of Ct comparison, and the expression level of the gene was normalized to that of the housekeeping gene β-actin. Table S1 lists the primers for the genes of interest.
Superscript 3 reverse transcriptase kit
Manufactured by Bio-Rad
Sourced in United States
The Superscript™ III Reverse Transcriptase Kit is a laboratory product designed for the synthesis of first-strand cDNA from RNA templates. The kit includes the Superscript™ III Reverse Transcriptase enzyme, which catalyzes the conversion of RNA into complementary DNA (cDNA) molecules.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green supermix, by Bio-Rad (3 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (3 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Spectrometer, by PerkinElmer (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Ddpcr system, by Bio-Rad (1 mentions)
Taqman copy number assay probes, by Bio-Rad (1 mentions)
Taqman probe, by Bio-Rad (1 mentions)
Ssoadvanced preamp supermix, by Bio-Rad (1 mentions)
Rneasy lipid tissue mini kit, by Qiagen (1 mentions)
Iq5 real time pcr detection system, by Bio-Rad (1 mentions)
6 protocols using superscript 3 reverse transcriptase kit
1
Quantitative Real-Time PCR Analysis of Skin Cytokines
2
Quantitative RT-PCR Analysis of Skin Gene Expression
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Total RNA was isolated from skin tissue using Trizol Reagent (Ambion Inc., Austin, TX, USA) reagent. The amount and purity of total RNA were assessed using a spectrometer 260 nm and 280 nm (Perkin Elmer, Boston, MA, USA). Each total RNA sample (1 µg) was used for synthesizing cDNA using the Superscript III reverse transcriptase kit (Bio-Rad, Richmond, CA, USA) that synthesizes each equal amount of total RNA into cDNA. The cDNA was mixed with SYBR Green supermix (Bio-Rad, Richmond, CA, USA) mixture and then amplified using CFX Connect™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc, Hercules, CA, USA). To evaluate the relative mRNA expression of the gene of interest in the skin using the Ct comparison method, the expression level of the genes was normalized to the expression of the housekeeping gene β-actin. Use the following primers designed and synthesized in the house for PCR reactions, mouse β-actin forward 5′-TCCTGTGGCATCCACGAAA CT-3′ and reverse 5′-GAAGCATTTGCGGTGGACGAT-3′. IL-4, forward 5′-GAATGTACCAGG AGCCATATC-3′ and reverse 5′-CTCAGTACTACGAGTAATCCA-3′. IL-13, forward 5′- CAGCT CCCTGGTTCTCTCAC-3′and reverse5′-CCACACTCCATACCATGCTG-3′. TNF-α, forward 5′-CCTGTAGCCCACGTCGTAGC-3′ and reverse5′-TTGACCTCAGCGCTGAGTTG-3′. The relative expression of each gene was evaluated by the Ct method [32 (link)].
3
Hippocampal RNA Extraction and qRT-PCR Analysis
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Hippocampal tissue (n = 5) was lysed with Trizol (Ambion Inc., Austin, TX, USA) reagent solution, and the total RNA was isolated. The amount and purity of the total RNA were determined at 260 nm and 280 nm using a spectrometer (Perkin Elmer, Boston, MA, USA). According to the manufacturer’s instructions, the total RNA (1 µg) was synthesized into cDNA using the Superscript™ III Reverse Transcriptase Kit (Bio-Rad, Richmond, CA, USA). The synthesized cDNA was mixed with SYBR Green supermix (Bio-Rad, Richmond, CA, USA) and amplified using the CFX Connect™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The relative mRNA expression of the ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) in the hippocampus was assessed using the cycle of threshold (CT) method. The gene expression levels were normalized with those of the housekeeping gene β-actin. The primers for the gene were as follows: β-actin: Forward: 5′-AGCGTGGCTACAGCTTCACC-3′, Reverse: 5′-AAGTCTAGGGCAACATAGCACAGC-3′. BDNF: Forward: 5′-ATGCCGAACTACCCAATCGT-3′, Reverse: 5′-GCCAATTCTCTTTTTGCTATCCA-3′. CNTF: Forward: 5′-ATGGCTTTCGCAGAGCAATCACC-3′, Reverse; 5′-GTCGTGTACTCTCGGTAATACC-3′.
4
ddPCR Quantification of RNA Expression
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RNA was extracted using the Rneasy Lipid tissue mini kit (QIAGEN, Hilden, Germany). cDNA was then generated using the Superscript III Reverse Transcriptase kit and pre-amplified with a mixture of the tested TaqMan® probes (Key Resources Table ) using the SsoAdvanced PreAmp Supermix (Biorad). Differential levels of cDNA expression were measured using the droplet digital PCR (ddPCR) system (Biorad) and TaqMan® copy number assay probes. Briefly, cDNA and 6-carboxyfluorescein (FAM) probes and primers were distributed into approximately 10,000 to 20,000 droplets. The nucleic acids were then PCR-amplified in a thermal cycler and read (as the number of positive and negative droplets) with a QX200 Droplet Digital PCR System. The ratio for each tested gene was normalized against the total number of positive droplets against 45S RNA.
5
Quantitative Analysis of Inflammatory Markers in Sepsis
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Total RNA samples were isolated from intestine muscularis using TRIzol reagent (Invitrogen, Thermo Fisher Scientific). Briefly, separating intestine muscularis and using liquid nitrogen quick freezing, then extracted with ethanol, isopropanol, and chloroform. A Thermo Fisher Scientific Superscript III Reverse Transcriptase kit was used to create cDNA, and RT-PCR was performed using a Bio-Rad machine. The data were subjected to relative quantification using 18S ribosomal RNA (2–ΔΔCt) as a control. Each sample was analyzed thrice. A list of RT-qPCR primers used is provided in Table 1.
6
Transcriptome Analysis of B. subtilis
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Strains were grown in LB medium supplemented with 1.0% D-glu- cose and 0.1 mM IPTG until an OD 600 of 1.0 was reached and 5 ml samples were harvested by centrifugation (7000 rpm, 4 °C). The cell pellets were immediately shock frozen in liquid nitrogen. The total RNA was extracted as described earlier (Yi et al., 2017) and split into two aliquots for RNA sequencing and qRT-PCR. Sequencing of cDNA versions of the mRNAs was accomplished by PrimBio (USA), and the data analysis was performed as described before (de Jong et al., 2015; van der Meulen et al., 2016) . The transcriptome of each sample was measured once, and for making the dispersion model in edgeR (Robinson et al., 2001) , we used two biological replicates of the WT B. subtilis 168 grown under identical conditions. For qRT-PCR, reverse transcription of the RNA samples was performed with the SuperScript™ III Reverse Transcriptase kit, and quantitative PCR analysis was done with the iQ5 Real-Time PCR Detection System (Bio-Rad) as described previously (Livak and Schmittgen, 2001) . Each experiment was performed in biological duplicate, and the mean value from three independent experiments was calculated for analysis.
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