Total cellular RNA was isolated from PHH cultured in 96-well plates using an RNeasy 96 Kit (Qiagen) following the manufacturer’s instructions. Real-Time RT-PCR was performed with TaqMan® Fast Virus 1-Step Master Mix (Life Technologies) using a QuantStudio 7 Flex Real-Time PCR System (Life Technologies) following the manufacturer’s instructions. β-actin mRNA expression was used to normalize target gene expression. All oligonucleotide primer sets were manufactured by Life Technologies.
Rneasy 96 kit
Manufactured by Qiagen
Sourced in Germany, United States, Switzerland, Spain
The RNeasy 96 kit is a laboratory equipment product manufactured by Qiagen. It is designed for the purification of total RNA from a variety of sample types. The kit utilizes a silica-membrane-based technology to efficiently capture and purify RNA molecules.
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Lab products found in correlation
Rlt buffer, by Qiagen (9 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (9 mentions)
Tissuelyser 2, by Qiagen (7 mentions)
Rneasy mini kit, by Qiagen (6 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
Dnase 1, by Thermo Fisher Scientific (5 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (5 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (4 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (4 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (4 mentions)
T100 thermal cycler, by Bio-Rad (4 mentions)
Takyon dry one step rt qpcr, by Eurogentec (3 mentions)
Magna lyser, by Roche (3 mentions)
Qiaamp vrna mini kit, by Qiagen (3 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (3 mentions)
Bullet blender, by Next Advance (3 mentions)
Quantitect probe rt pcr kit, by Qiagen (3 mentions)
Truseq stranded mrna library preparation kit, by Illumina (3 mentions)
124 protocols using rneasy 96 kit
1
Quantitative RT-PCR for mRNA Analysis
2
RSV RNP Complex Transcription Assay
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Transcription reactions contained 25 μg of crude RSV RNP complexes in 30 μL of reaction buffer (50 mM Tris-acetate (pH 8.0), 120 mM potassium acetate, 5% glycerol, 4.5 mM MgCl2, 3 mM DTT, 2 mM EGTA, 50 μg ml−1 BSA, 2.5 U RNasin, 20 μM ATP, 100 μM GTP, 100 μM UTP, 100 μM CTP, and 1.5 μCi [α-32P]ATP (3,000 Ci mmol−1)). The radiolabelled nucleotide used in the transcription assay was selected to match the nucleotide analogue being evaluated for inhibition of RSV RNP transcription.
To determine whether nucleotide analogues inhibited RSV RNP transcription, compounds were added using a six-step serial dilution in fivefold increments. After a 90-min incubation at 30 °C, the RNP reactions were stopped with 350 μl of Qiagen RLT lysis buffer, and the RNA was purified using a Qiagen RNeasy 96 kit. Purified RNA was denatured in RNA sample loading buffer at 65 °C for 10 min and run on a 1.2% agarose/MOPS gel containing 2 M formaldehyde. The agarose gel was dried, exposed to a Storm phosphorimaging screen, and developed using a Storm phosphorimager.
To determine whether nucleotide analogues inhibited RSV RNP transcription, compounds were added using a six-step serial dilution in fivefold increments. After a 90-min incubation at 30 °C, the RNP reactions were stopped with 350 μl of Qiagen RLT lysis buffer, and the RNA was purified using a Qiagen RNeasy 96 kit. Purified RNA was denatured in RNA sample loading buffer at 65 °C for 10 min and run on a 1.2% agarose/MOPS gel containing 2 M formaldehyde. The agarose gel was dried, exposed to a Storm phosphorimaging screen, and developed using a Storm phosphorimager.
3
T Cell-Mediated Inhibition of SARS-CoV-2 Replication
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EBV-transformed BCLs expressing ACE2 were infected with SARS-CoV-2 viruses at an MOI of 0.1 for 120 min at 37 °C. Cells were washed and cocultured with T cells at an E:T ratio of 4:1. Control wells containing virus-infected targets without T cells were also included. After 48 h incubation, cells were washed with PBS and lysed with buffer RLT (QIAGEN). RNA was extracted using RNeasy 96 kit (QIAGEN). Virus copies were quantified with Takyon Dry one-step RT-qPCR (Eurogentec) using SARS-CoV-2 (2019-nCoV) CDC qPCR Probe Assay (IDT, ISO 13485:2016) and human β2-microglobulin as an endogenous control (Applied Biosystems). The suppression rate was calculated by the percentage reduction of virus replication by T cells.
4
Quantifying ZIKV RNA Levels in Mice
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ZIKV-infected mice were euthanized using a ketamine/xylazine cocktail on the indicated days post-infection, and blood and organs were collected. Serum was separated from coagulated blood, and tissues were weighed and homogenized with beads using a MAgNA Lyser (Roche). RNA was extracted using RNeasy Mini Kit or RNeasy 96 kit (QIAGEN). Viral RNA levels were measured using TaqMan one-step quantitative reverse transcriptase PCR (RT-qPCR) on an ABI 7500, after comparison with a standard curve generated using 10-fold serial dilution of viral RNA from known infectious virus quantities. Viral burdens were expressed as viral RNA equivalents per gram or milliliter on a log10 scale. For RT-qPCR, published ZIKV primers and probe set were used (Richner et al., 2017a ): Forward 5′-CCACCAATGTTCTCTTGCAGACATATTG-3′; Reverse 5′-TTCGGACAGCCGTTGTCCAACACAAG-3′; Probe 5′-/56 FAM/AGCCTACCT/ZEN/TGACAAGCAGTC/3IABkFQ/−3′ (Integrated DNA Technologies).
5
Quantitative RT-PCR for Gene Knockdown
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Total RNA was harvested from cells using RNeasy 96 kit (Qiagen) for measuring KD efficiency upon shRNA transduction. Cellular RNAs were reverse transcribed, and PCR amplified using the SuperScript III Platinum One-Step qRT-PCR System with Platinum Taq (Invitrogen) and IDT Primer Assays (Integrated DNA Technologies). Cellular RNAs were normalized to 18S levels using StepOnePlus System (Applied Biosystems).
6
Gene Expression Analysis of Inflammatory and Remodeling Markers
7
Macrophage Isolation and Gene Expression
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Following the application of macrophages to excised skin wounds, tissue was harvested from the delivery site 3 days after surgery, minced, and digested using collagenase type II and collagenase type IV for 1 h at 37°C. To stop the reaction, cells were centrifuged and resuspended as a single‐cell suspension in buffer (PBS with 1% BSA). GFP+ cells were sorted as single cells using a FACsAria cytometer (BD Biosciences). Cultured macrophages were sorted as a control population. Total RNA from transplanted cells was isolated using the RNeasy 96 Kit (QIAGEN) following the manufacturer's instructions. Then, reverse transcribed into cDNA using GoScript™ Reverse Transcriptase according to the manufacturer's instructions (Promega). Quantitative real‐time PCR analysis was performed using a GoTaq® qPCR Master Mix Kit (Promega) in an ABI 7300 Fast Real‐time PCR System (Applied Biosystems, FosterCity, CA). The relative mRNA expression level of each gene was normalized with the β‐actin in the same sample.
8
RNA Extraction and Real-Time RT-PCR
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RNA was extracted from 50 μl NW or 0.1-g tissue homogenates using QIAamp vRNA mini kit or the RNeasy 96 kit (QIAGEN) and eluted with 60 μl of water. 5 μL RNA was used for each reaction in real-time RT-PCR.
9
Quantitative RNA Analysis in Mouse Tissues
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Cultured cells were lysed in 300 μl of RLT buffer (Qiagen) containing 1% (v/v) 2-mercaptoethanol (BME, MilliporeSigma). “For RNA extraction, the following were dissected: (a) a 2-mm coronal section of the cortex at 1 mm posterior to the injection site, (b) the hippocampus, and (c) a 2-mm coronal section of the thoracic spinal cord. Tissues were homogenized in 500 μl of RLT buffer containing 1% (v/v) BME. RNA was isolated from 20 μl of lysate with an RNeasy 96 Kit (Qiagen) that included in-column DNA digestion with 50 U of DNase I (Invitrogen). RT-PCR was performed using StepOne Realtime PCR system (Applied Biosystems), as described previously (50 (link)). The sequences of primers and probes are provided in Supplemental Table 1 . PCR results were normalized by housekeeping gene cyclophilinA/Ppia and further normalized to the level in PBS-treated mice or untreated cells.
10
Viral Protease Quantification by RT-PCR
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At 48 h after transfection of 293Ts, RNA was prepped according to the RNeasy 96 kit (Qiagen). One-step RT-PCR was performed using primers targeting the respective CoV 3CLpro (SARS-CoV-2, SARS-CoV, MHV, IBV, or 229E) or PR8 NP and a housekeeping gene (18S) with the iTaq universal SYBR green one-step kit (Bio-Rad) on an Applied Biosystems QuantStudio3 instrument.
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