RNA was extracted from whole blood, plasma, and tissue using RNeasy Protect Animal Blood, QIAamp Viral RNA Mini, and RNeasy Mini kits (Qiagen). Tissue samples were homogenized using a FastPrep®-24 homogenizer and Lysing Matrix D (MP Biomedicals). qPCR primers and probes ZIKV_1086, ZIKV_1162c, ZIKV_1107_P were synthesized by Integrated DNA Technologies [48 (link)]. Viral loads were quantified by qRT-PCR using the SuperScript™ III Platinum™ One-Step qRT-PCR Kit (Thermo Fisher) and a LightCycler 480 (Roche) instrument as previously described [10 (link)]. To determine the concentration of ZIKV RNA in a sample, a synthetic DNA fragment corresponding to the H/PF/2013 genome from positions 1086 to 1107 was amplified and subcloned into pCR4-TOPO (Thermo Fisher). RNA standards were synthesized using a MAXIscript™ T7/T3 Transcription Kit (Thermo Fisher), quantified by absorbance and Quant-iT Ribogreen RNA Assay Kit (Life Technologies), and serially diluted to generate in-run standard curves for qRT-PCR analysis. To quantify ZIKV NS1-antigenemia, plasma from guinea pigs was subjected to a Zika Virus NS1 ELISA (BioFront Technologies).
Maxiscript t7 t3 transcription kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MAXIscript™ T7/T3 Transcription Kit is a laboratory tool used for the in vitro synthesis of RNA from DNA templates. It contains the necessary reagents, including RNA polymerases, nucleotides, and buffers, to facilitate the transcription process.
Automatically generated - may contain errors
Lab products found in correlation
Biotin rna labeling mix, by Roche (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Pcr4 topo, by Thermo Fisher Scientific (1 mentions)
Qiaamp viral rna mini, by Qiagen (1 mentions)
Fastprep 24 homogenizer, by MP Biomedicals (1 mentions)
Lysing matrix d, by MP Biomedicals (1 mentions)
Superscript 3 platinum one step qrt pcr kit, by Thermo Fisher Scientific (1 mentions)
Quant it ribogreen rna assay kit, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Rnase r, by Illumina (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Biotin rna labelling mix, by Roche (1 mentions)
Pierce magnetic rna protein pull down kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using maxiscript t7 t3 transcription kit
1
Quantification of Zika Virus Infection
2
Circular RNA Synthesis from Linear RNA
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Linear RNAs were transcribed using MAXIscript™ T7/T3 Transcription Kit (Thermo Scientific) and Biotin RNA Labeling Mix (Roche) according to the manufacturer’s instructions. Linear RNAs were incubated with the DNA splints (molar ratio = 1:1.5) at 95 °C for 5 min in the annealing buffer (100 mM NaCl, 10 mM Tris-HCl pH 7.5), and then cooled down to room temperature and ligated with T4 DNA ligase (NEB) at 16 °C overnight to form circRNAs. After treatment with RNase R (Epicentre) and DNase I (Thermo Scientific) at 37 °C for 20 min to remove DNA templates and linear transcripts, circRNAs were purified by phenol-chloroform extraction. The primers and DNA splints are shown in Supplementary Table 5 .
3
Biotinylated Atrolnc-1 RNA Pulldown
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To obtain the biotin‐labelled Atrolnc‐1‐RNA probe, linearized pBlue‐Atrolnc‐1 plasmid was incubated with biotin RNA labelling mix (Roche Molecular Systems, Inc. Hague Road, IN) for 20 min at 37°C, in vitro transcription was performed using a MAXIscript® T7/T3 Transcription Kit (Thermo Fisher, Waltham, MA, USA) following manufacturer's instructions. Biotinylated Atrolnc‐1‐ RNA was incubated with RNA structure buffer at 95°C for 2 min, on ice for 3 min, at room temperature for another 30 min to allow proper RNA secondary structure formation. The biotinylated Atrolnc‐1‐RNA probe was added into prewashed hydrophilic streptavidin magnetic beads and incubated at 4°C with gentle rotation for overnight. C2C12 cytoplasmic lysate and nuclear lysate were prepared and incubated with magnetic beads for 2 h at 4°C with rotation. Wash the magnetic beads three times, resuspend the beads and boil beads with 1× loading buffer at 95°C for 10 min, proteins were separated by SDS‐PAGE. The gels were stained with 0.1% Coomassie Brilliant Blue R250 for 1 h.
4
Investigating MIAT lncRNA Protein Interactions
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Briefly, MIAT and its antisense RNA were chemically synthesized and were inserted into the sites between KpnI and SacI in pBluescript II SK+. The sense and antisense MIAT were transcribed in vitro using MAXIscript T7/T3 Transcription Kit (Thermo Fisher Scientific, Carlsbad, CA, USA). Then, the lncRNAs were labelled with biotin using a Biotin RNA Labeling Mix (Roche, Germany) and purified using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Then, cell lysate from primary rat neurons with EGLN2 overexpression was prepared. RNA pull‐down assay was performed using a Pierce Magnetic RNA‐Protein Pull‐Down Kit (Thermo Fisher Scientific), according to the manufacturer's instruction. The retrieved protein was eluted from the RNA‐protein complex and was subjected to SDS‐PAGE. Sense MIAT‐specific gel bands were excised and trypsin digested. Then, the peptides were analysed by liquid chromatography tandem mass spectroscopy (LC‐MS/MS), following the method introduced previously.16
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