rAAV-DJ were produced in HEK293T cells (ATCC) by triple transient transfection using polyethylenimine (PEI, Polysciences Inc). For each vector, fourteen 15 cm dishes were transfected at 80% confluency with pAd5 (helper plasmid), rAAV-DJ genome plasmid and vector plasmid at a 3:1:1 ratio4 . In total, each plate was transfected with 41.25 μg of DNA. Purification was performed with AAVpro Extraction Kit (Takara) according to the manufacturer protocol. Titer quantification was performed by qPCR using SYBRGreen (PCR Biosystems). A list of primers used for AAV titer quantification can be found in Supplementary Table 1 .
Sybrgreen
Manufactured by PCR Biosystems
Sourced in United Kingdom
SYBRGreen is a fluorescent dye used in real-time PCR (qPCR) assays. It binds to double-stranded DNA and emits fluorescence upon excitation, allowing the monitoring and quantification of DNA amplification during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Hek293t cell, by American Type Culture Collection (2 mentions)
Polyethylenimine (pei), by Polysciences (2 mentions)
Aavpro extraction kit, by Takara Bio (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Steponeplus qpcr system, by Thermo Fisher Scientific (2 mentions)
Rq1 dnase, by Promega (2 mentions)
Primestar max, by Takara Bio (2 mentions)
Dneasy blood tissue kit, by Qiagen (2 mentions)
Ampure xp bead, by Beckman Coulter (2 mentions)
Reliaprep rna tissue miniprep system, by Promega (2 mentions)
Rneasy kit, by Qiagen (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Lightcycler 96, by Roche (1 mentions)
Takyon one step rt probe mastermix, by Eurogentec (1 mentions)
Cfx96, by Bio-Rad (1 mentions)
Rna extraction kit, by Macherey-Nagel (1 mentions)
Cdna synthesis kit, by Quanta Biosciences (1 mentions)
Lysis buffer, by Macherey-Nagel (1 mentions)
Viia 7 system, by Thermo Fisher Scientific (1 mentions)
Steponeplus system, by Thermo Fisher Scientific (1 mentions)
9 protocols using sybrgreen
1
Production and Quantification of rAAV-DJ
2
Quantifying Donor AAV and Cas9 in Tissues
3
Quantification of SARS-CoV-2 and Cellular RNA
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Total cellular RNA was extracted using the RNeasy kit (Qiagen) or Trizol (Thermofisher) according to manufacturer’s instructions. For quantification of viral or cellular RNA, equal amounts of RNA, as determined by nanodrop, were used in a one-step RT-qPCR using the Takyon-One Step RT probe mastermix (Eurogentec) and run on a Roche Light Cycler 96. For quantification of viral copy numbers, qPCR runs contained serial dilutions of viral RNA standards. Total SARS-CoV-2 RNA was quantified using: 2019-nCoV_N1-F: 5’-GAC CCC AAA ATC AGC GAA AT-3’, 2019-nCoV_N1-R: 5’-TCT GGT TAC TGC CAG TTG AA TCT G-3’, 2019-nCoV_N1-Probe: 5’-FAM-ACC CCG CAT TAC GTT TGG TGG ACC-BHQ1-3’. SOAT1 and SOAT2 RNA was quantified by SYBR green (PCR Biosystems) using the following primers, SOAT1_F: 5’-GCT CGT GTT CTG GTC CTA TGT G-3’, SOAT1_R: 5’-TAG AAC ATC CTG TCA CCA AAG CG-3’, SOAT2_F: 5’-CAA TGG ACC CGA CAC ATG GA-3’, SOAT2_R: 5’-GGG GCA GAG GTT TGT CTT GT-3’. These were expressed relative to the housekeeper gene Beta-2-Microglobin using B2M_F: 5’-CTA CAC TGA ATT CAC CCC CAC TG-3’ and B2M_R: 5’-ACC TCC ATG ATG CTG CTT ACA TG-3’.
4
Zebrafish RNA Expression Analysis
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For each exposure group, 8 biological replicates were used and for each biological replicate, 4 larvae at 6 dpf were pooled in a homogenizing tube. The samples were lysed in lysis buffer (Macherey Nagel) and RNA extraction was performed using RNA extraction kit (Macherey Nagel). cDNA was prepared using the cDNA synthesis kit (Quanta Biosciences). The qPCR was carried out on thermocycler (CFX96; BioRad) using SYBR Green (PCR Biosystems). The qPCR cycles consisted of an initial denaturation at 95 °C for 2 min, 40 cycles of 95 °C for 5 s and 60 °C for 30s. The data was normalized using elongation factor (eef1a1) and ΔΔCt method was used to calculate fold change (Schmittgen and Livak, 2008 (link)). Primer sequences are listed in Table S1.
5
RNA Extraction and Gene Expression Analysis
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Total RNA was extracted using the ReliaPrep™ RNA Tissue miniprep system (Promega). For the mouse cell lineage identification, RT2 profiler PCR array (Qiagen), triplicate samples were pooled and 400 ng RNA reverse transcribed with M-MLV RT (Promega). Relative gene expression using SYBR green (PCR Biosystems) was performed on a ViiA™ 7 system (Applied Biosystems) and quantified using the comparative Ct method. β-Actin was selected for normalization. For other analyses, 1 μg RNA was reverse transcribed and quantified on a StepOnePlus™ system (Applied Biosystems). Canx was used for normalisation, selected using the geNorm™ reference selection kit (Primerdesign). qPCR primer sequences are shown in Table 1 .
6
Colon RNA Extraction and qPCR
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Colon tissue was flash frozen in LN2 and stored at −70 °C until use. Tissue (∼20 mg) was homogenised in QIAzol lysis buffer (Qiagen, Hilden, Germany) and 200 µl of chloroform was added before 15 s vigorous mixing. Phases were then separated by centrifugation at 12,000 rpm for 15 min at 4 °C after which the upper aqueous phase was taken off and transferred to a separate tube. RNA was purified using an RNeasy spin column kit (Qiagen) following the manufacturer’s instructions. RNA concentration and purity was assessed using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham,MA, USA) checking that the A260/A280 and the A260/230 ratios were both approximately 2.0 indicating low protein and organic compound contamination, respectively. RNA was reverse transcribed using SuperScript II (Thermo Fisher Scientific) as per the manufacturer’s instructions. cDNA samples were stored at −20 °C until used, Gene expression using cDNA diluted to 5 ng/µl was assayed using Primer Design PrecisionPlus Master Mix with premixed SYBR Green (PCR BioSystems, London, UK), using a MX3000P qPCR system (Stratagene, San Diego, USA). Gene expression data were normalized to 18S and analysed using the ΔΔCt method [35] (link). Oligonucleotide primers were obtained from Sigma-Aldrich or Primer Design (Supplementary Table 2 ).
7
Quantitative RT-PCR for Mouse Heart
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Total RNA was extracted from mouse hearts using ReliaPrep™ RNA Tissue miniprep system (Promega). cDNA was synthesized using M-MLV Reverse Transcriptase (Promega). Primers for real-time polymerase chain reaction (RT-PCR) are listed in Supplementary material online , Table S2 . RT-PCR reactions were performed using SYBR-Green (PCR biosystems) in a StepOnePlus Real-time PCR System (Applied Biosystems).
8
Production and Quantification of rAAV-DJ
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rAAV-DJ were produced in HEK293T cells (ATCC) by triple transient transfection using polyethylenimine (PEI, Polysciences Inc). For each vector, fourteen 15 cm dishes were transfected at 80% confluency with pAd5 (helper plasmid), rAAV-DJ genome plasmid and vector plasmid at a 3:1:1 ratio4 . In total, each plate was transfected with 41.25 μg of DNA. Purification was performed with AAVpro Extraction Kit (Takara) according to the manufacturer protocol. Titer quantification was performed by qPCR using SYBRGreen (PCR Biosystems). A list of primers used for AAV titer quantification can be found in Supplementary Table 1 .
9
Quantifying Donor AAV and Cas9 in Tissues
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For copy number quantification of the donor AAV in tissue samples, DNA was extracted from fresh tissues using DNeasy Blood & Tissue Kit (Qiagen) with RNAse treatment on-column. Each sample was analyzed for both internal control (Albumin intron2 ) and the donor AAV. For quantification of donor AAV copy number per haploid genome, a standard curve was used. Standards were prepared from a PCR PrimeStar MAX (Takara) reaction using naïve C57BL/6OlaHsd mice genomic DNA for the internal control or donor AAV plasmid for the Donor sample and purified using AMPure XP beads (Beckman Coulter) at a 1:1 ratio. A list of primers used for Donor and internal control reactions can be found in Supplementary Table 1 . Standard curve amplicons were quantified using Qubit (Invitrogen) and serially diluted 8 times. For AAV Cas9 quantification RNA was extracted from fresh tissues using RNeasy Mini Kit (Qiagen) with DNAse treatment on-column and post-purification using RQ1 DNAse (Promega). Reverse transcription was performed using RevertAid (ThermoFisher) and random hexamer primers. Data collection and analysis were performed on a StepOnePlus qPCR System (Applied Biosystems) using SYBRGreen (PCR Biosystems). For fold change of AAV titers and Cas9 relative expression, we used the relative quantity method13 .
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