Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was performed on RNA obtained from harvested ear skin. Complementary DNA (cDNA) was generated using the Superscript III Reverse Transcriptase First Strand Synthesis System (Invitrogen) using the manufacturer’s protocol with random hexamers. Samples were pre-amplified using pooled Taqman assays (ABI) and PreAmp Master Mix (Fluidigm) according to the Gene Expression Preamplification with Fluidigm PreAmp Master Mix and Taqman assays protocol (Fluidigm). Expression of 48 control and inflammatory genes was assessed using a 48x48 qPCR dynamic array (Fluidigm), with a panel of Taqman assays (ABI), according to manufacturer’s instructions. Data were calculated by the ΔΔCT method, using either GAPDH or GUSB in each experiment to normalize transcripts within samples. Each normalized ΔCT value was compared to the average ΔCT for the same transcript in sham injected mice to get the -ΔΔCT, yielding the log2(fold change).
Superscript 3 reverse transcriptase first strand synthesis system
Manufactured by Thermo Fisher Scientific
Sourced in United States
Superscript III Reverse Transcriptase First Strand Synthesis System is a lab equipment product by Thermo Fisher Scientific. It is designed for reverse transcription of RNA to synthesize complementary DNA (cDNA) strands.
Automatically generated - may contain errors
Lab products found in correlation
Taqman assays, by Standard BioTools (1 mentions)
Fluidigm preamp master mix, by Standard BioTools (1 mentions)
Preamp master mix, by Standard BioTools (1 mentions)
Vector nti version 11, by Thermo Fisher Scientific (1 mentions)
Q5 high fidelity polymerase, by New England Biolabs (1 mentions)
Zr viral rna kit, by Zymo Research (1 mentions)
Ex taq dna polymerase, by Takara Bio (1 mentions)
Qiaamp viral rna mini kit, by Qiagen (1 mentions)
Nucleospin rna 2 kit, by Macherey-Nagel (1 mentions)
Abi prism 7900ht, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
4 protocols using superscript 3 reverse transcriptase first strand synthesis system
1
Quantitative RNA Expression Analysis in Skin
2
Virus Genome Sequencing Protocol
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For all viruses generated, the entire genome was sequenced from the cDNA clone. In addition, the entire structural poly-protein was sequenced for all virus stocks used in all experiments, maintained 100% match with cDNA sequence. Sequencing was performed by first extracting viral RNA using the ZR Viral RNA kit (Zymo Research, Irvine, CA). Reverse-transcription was then performed to produce cDNA using the Superscript III Reverse-Transcriptase First-Strand Synthesis System (Invitrogen, Carlsbad, CA). cDNA was then used as a template for PCR with virus-specific primers using Q5 high fidelity polymerase (New England Biolabs, Ipswich, MA). The amplicons were then used for sequencing at the Biotech Center located at the University of Wisconsin-Madison. Vector NTI (version 11.5, Invitrogen) was used to align and assemble sequencing data.
3
RNA Extraction and cDNA Synthesis
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QIAamp Viral RNA Mini Kits were purchased from QIAGEN GmbH (Hilden, Germany). SuperScript III First-Strand Synthesis System reverse transcriptase was purchased from Invitrogen Inc. (Carlsbad, CA, United States). ExTaqDNA polymerase was purchased from Takara Biotechnology (Dalian) Co., Ltd. (China).
4
Quantitative Real-Time PCR Analysis of Zika Virus in Monocytes
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Total RNA was extracted from monocytes with the NucleoSpin RNA II kit (Macherey-Nagel). First-strand complementary DNA synthesis was performed with the SuperScript III First-Strand Synthesis System Reverse Transcriptase (Invitrogen). Quantitative real-time PCR was performed on a real-time PCR system (ABI PRISM 7900HT) with SYBR Green PCR Master Mix (Life Technologies). Data were analyzed with the 2-DDCT method75 (link), with all samples normalized to GAPDH. The ZIKV forward and reverse primers used were 5′-AARTACACATACCARAACAAAGTGGT-3′ and 5′-TCCRCTCCCYCTYTGGTCTTG-3′76 (link). The GAPDH forward and reverse primers used were 5′-GGTCGGAGTCAACGGATTTG-3′ and 5′- ACTCCACGACGTACTCAGCG-3′. The CD99 forward and reverse primers used were 5′-CTCTTCCCCTTCTTTCCTGTG-3′ and 5′-CAAATCCAAACCCCAACCAC-3′. The ITGAL forward and reverse primers used were 5′- TCATACACCACGTCAACCTTC-3′ and 5′-CTCTTCCATGTTCAGCCTCTG-3′.
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