Following harvest at time points described in the corresponding figure legend, lungs were snap frozen and stored at -80°C until use. Total RNA was extracted using TRIzol reagent (Life Technologies) per the company's protocol. 1 μg of RNA was subjected to reverse transcription using SuperScript III RNase reverse transcriptase (Life Technologies, San Diego, CA) to create cDNA. Real-time quantitative polymerase chain reaction (PCR) was performed using SYBR green master mix (Applied Biosystems, San Francisco, CA). We measured tumor necrosis factor- (TNF-) α, interleukin- (IL-) 1β, IL-6, IL-10, IL-12p35, IL-12p40, interferon- (IFN-) γ, and keratinocyte chemoattractant (KC). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene. The sequences of primers are listed in Supplementary Table 1 .
Superscript 3 rnase reverse transcriptase
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Superscript III RNase reverse transcriptase is a thermostable enzyme used for the synthesis of first-strand cDNA from RNA templates. It exhibits reduced RNase H activity and increased thermal stability compared to other reverse transcriptases.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green master mix, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
S1000 thermal cycler, by Bio-Rad (1 mentions)
Sybr green pcr core reagents system, by Thermo Fisher Scientific (1 mentions)
Nucleospin rna 2, by Macherey-Nagel (1 mentions)
Steponeplus system, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
6 protocols using superscript 3 rnase reverse transcriptase
1
Inflammatory Cytokine Expression Analysis
2
Quantitative gene expression analysis
3
Reverse Transcription and qRT-PCR for Gene Expression Analysis
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First-strand cDNA synthesis was performed using approximately 1 μg of total RNA, oligo dT primers and Superscript III RNase reverse transcriptase (Invitrogen). Synthesized cDNA was used for PCR amplification with the specific primers listed in Supplementary Table 1 . All PCRs were carried out in a S1000TM thermal cycler (Bio-Rad) with the following parameters: [30 (link)–35 (link)] cycles of 30 sec at 95°C, 30 sec at 55°C and 30 sec at 72°C. Quantitative reverse-transcription-PCR (qRT-PCR) was performed to analyze transcripts identified by microarray assays. The SYBR Green PCR Core Reagents system (Applied Biosystems, Foster City, CA) was used for real-time monitoring of amplification.
4
Quantifying RNA Expression in PBMC and Placenta
5
Quantitative RT-PCR Analysis of UBE2C
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Total RNA was extracted with TRIzol reagent (Biocompare, South San Francisco, CA, USA). A total of 1 μg of RNA was reverse-transcribed with SuperScriptIII RNase Reverse Transcriptase (Invitrogen-GIBCO, Carlsbad, CA, USA) for cDNA synthesis. Real-time polymerase chain reaction (PCR) was carried out using a StepOnePlusTM system (Applied Biosystems, Foster City, CA, USA) with SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA). All genes expression levels in cells was normalized with internal control GAPDH gene. The UBE2C primers sequences were shown as follows—the forward—5′-TGATGTCTGGCCATAAAGGGA-3′, the reverse—5′-AGCGAGAGCTTATACCTCAGG-3′.
6
Macrophage RNA Isolation and cDNA Synthesis
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Macrophage-like cells from the head kidney were isolated and treated with LPS and curdlan 48 hours before harvested. The cells were lysed in a RT-buffer containing 2mercaptoethanol and kept at -80℃. RNA was isolated using RNeasy Mini Kit by Qiagen (Germany) -according to the manufacturer's guidelines. The yield and purity of the RNA was determined using a NanoDrop (Nano-Drop Technologies, Wilmington, DE, USA). The samples having OD 260/280 values between 1.9 and 2.1 was processed further.
To avoid the risk of having contaminating DNA, interfering with the assay, in the samples -the samples were pre-treated with DNase I (1 U µg -1 RNA; Invitrogen, USA).
To synthesize first-strand cDNA, a SuperScript III RNase reverse transcriptase (Invitrogen) was used, as described by Kumari et al. (2015) (link).
To avoid the risk of having contaminating DNA, interfering with the assay, in the samples -the samples were pre-treated with DNase I (1 U µg -1 RNA; Invitrogen, USA).
To synthesize first-strand cDNA, a SuperScript III RNase reverse transcriptase (Invitrogen) was used, as described by Kumari et al. (2015) (link).
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