Total RNA was isolated from the heat inactivated nasopharyngeal swab samples using Direct-zolTM RNA Microprep (R2060, Zymo Research) by following the manufacturer’s instruction. In brief, 300 μL of nasopharyngeal swab samples were lysed in 400 μL of Trizol. Then 700 μL of 100% ethanol was added, followed by column purification using Zymo-SpinTM Column. Direct-zolTM RNA PreWash and RNA Wash Buffer were added sequentially to wash the column. Finally, RNA was eluted in 12 μL of nuclease free water and stored in −80°C until future use.
Direct zoltm rna microprep
Manufactured by Zymo Research
Sourced in United States
The Direct-zolTM RNA MicroPrep is a rapid and efficient method for the purification of total RNA from small samples, including cells, tissues, and other biological sources. It utilizes a spin column-based approach to isolate high-quality RNA suitable for downstream applications such as RT-qPCR, RNA sequencing, and other analyses.
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Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Viia real time pcr system, by Thermo Fisher Scientific (1 mentions)
Taqman reagent, by Thermo Fisher Scientific (1 mentions)
Revertaid reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (1 mentions)
Taqman fast universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Qiazol, by Qiagen (1 mentions)
Ncounter low rna input amplification kit, by NanoString (1 mentions)
Nextera xt dna library prep, by Illumina (1 mentions)
Smarter ultra low input rna kit for sequencing v4, by Takara Bio (1 mentions)
Nextseq 500, by Illumina (1 mentions)
7 protocols using direct zoltm rna microprep
1
RNA Extraction from Nasopharyngeal Swabs
2
RNA Extraction and qRT-PCR Analysis
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Total RNAs were extracted and purified with Direct-zolTM RNA MicroPrep (Zymo Research) according to manufacturer’s instructions under RNase free conditions. Five hundred ng of total RNA was reverse-transcribed using Revertaid first strand cDNA synthesis kit (Thermo Fisher Scientific). Real time quantification was performed in triplicate with ViiA Real-Time PCR system (ThermoFisher Scientific). Each sample was analyzed in triplicates. Expression of TBP (TATA-binding protein) was used as an internal loading control and 2-ΔΔ CT method was used for relative expression computation. Primer sequences are summarized in Table S1
3
Transcriptional Analysis of Zebrafish Embryos and HUVEC Cells
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RNA extraction was performed making use of Direct-zolTM RNA MicroPrep (Zymo Research, R2060, R2061, R2062, R2063). First strand cDNA synthesis was performed with Thermo Fisher Scientific RevertAid Reverse Transcriptase kit (Thermo Fisher Scientific, EP0441) with random hexamer primers for cells extracted from zebrafish embryos, and RNeasy plus Mini Kit (Qiagen, 74034) for HUVEC samples. SYBR Green real-time quantitative PCR was performed following SG qPCR Master mix protocol and reagents from Roboklon (EURx Roboklon, E0402-01) for zebrafish samples. Primers used for quantitative RT PCR were as previously published (Cvejic et al., 2008 (link)). For HUVEC samples, real-time quantitative PCR was performed using TaqMan reagents (Applied Biosystems).
4
Quantification of IL12 gene expression
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RNA was isolated using QIAzol (QIAGEN, Hilden, Germany) and Direct-zoltm RNA MicroPrep (Zymo Research, Irvine, CA, USA) from adherent cells according to the manufacturer’s instructions. Reverse transcription was performed using a High-Capacity RNA-to-cDNA Kit (Applied Biosystems-Thermo Fisher Scientific). Real-time PCR was performed using TaqMan® Fast Universal PCR Master Mix (Applied Biosystems-Thermo Fisher Scientific) and SDS 2.4 on a 7900HT Fast Real-Time PCR System (Applied Biosystems-Thermo Fisher Scientific), as previously described [31 (link)]. The following TaqMan® gene expression assay (Applied Biosystems-Thermo Fisher Scientific) was used in Real Time PCR analyses: IL12 (assay ID: Mm00434169_m1; best coverage assay to recognize interleukin 12a gene). The expression of this gene was normalized to B2m (assay ID: Mm00437762_m1). PCR data were analyzed using the 2-ΔCt method.
5
Purification and RNA Extraction for Transcriptome Analysis
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After purified by the TR-NanoVelcro CTC purification system, the purified cells were lysed, and the RNA was extracted using the Direct-zolTM RNA MicroPrep (Zymo Research, CA, USA) kit following the manufacturer's protocol. In brief, cells were lysed with 600 μL of Trizol solution and mixed with 600 μL of ethanol. The mixed solution was put in to Zymo-SpinTM IIC Column and centrifuged. After centrifugation the solution was washed twice with supplied wash buffer. The RNA was eluted from the column using 12 μL of RNA grade water. The amount of the eluted RNA needs to be above 2 ng/μL to pass the internal quality control. The RNA was then subjected to reverse transcription to convert to cDNA and whole transcriptome amplification using the nCounter Low RNA Input Amplification Kit (NanoString Technologies, Inc., WA, USA) following manufacturer's protocol.
6
Single-cell RNA Sequencing of Oocytes
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RNA isolation and purification were performed using Direct-zolTM RNA MicroPrep according with manufacturer instructions (Zymo Research, Irvine, CA) on three individual oocytes. Briefly, frozen eggs were thawed at room temperature and resuspended in Tri Reagent, followed by addition of 95% ethanol, DNase I treatment, and centrifugation in an IC column [34 (link)]. Final RNA elution was performed using DNase/RNase-free water. Total RNA was processed for library construction by Cofactor Genomics (St. Louis, MO) according to the following procedure: Briefly, total RNA was reverse-transcribed using an Oligo (dT) primer, and limited cDNA amplification was performed using the SMARTer® Ultra® Low Input RNA Kit for Sequencing – v4 (Takara Bio USA, Inc., Mountain View, CA). The resulting full-length cDNA was fragmented and tagged, followed by limited PCR enrichment to generate the final cDNA sequencing library (Nextera® XT DNA Library Prep, Illumina, San Diego, CA). Libraries were sequenced as single-end 75 base pair reads on an Illumina NextSeq500 following the manufacturer’s instructions.
7
Direct-zol RNA Microprep Protocol
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RNA isolation was performed using the Direct-zol TM RNA Microprep (Zymo Research Irvine, CA, USA) according to the manufacturer's instructions.
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