Total mRNA was isolated from cells with the RNeasy mini kit (Qiagen, Courtaboeuf, France) following the manufacturer’s instructions. RNA concentration and integrity were checked by optical density measurement at 230 nm and by 260/280 nm ratio (Thermo Scientific NanoDrop 1000, Labtech, Palaiseau, France). 1 μg of total mRNA was reverse transcribed using the high capacity cDNA reverse transcriptase kit (Applied Biosystems, Courtaboeuf, France) according to the manufacturer’s instructions. Polymerase chain reaction (PCR) assay was performed using the Sybr Green PCR Master Mix (Applied Biosystems) and selected primers (see Table 1 ) for the measurement of gene expression. Amplification and detection of PCR products were performed using Quant StudioTM 12 K Flex (Life Technologies). The relative expression ratio of each sample was normalized to the geometric mean of the expression level of Gapdh, Ppia and Rpl27 used as housekeeping genes61 (link).
Nanodrop 1000
Manufactured by EQT
Sourced in United Kingdom
The NanoDrop 1000 is a spectrophotometer designed for the quantification and analysis of small volume samples. It utilizes a patented sample retention technology that requires only 1-2 microliters of sample to perform measurements.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna blood mini kit, by Qiagen (1 mentions)
Microbead kits, by Miltenyi Biotec (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Myiq single color real time pcr detection system, by Bio-Rad (1 mentions)
Superscript 2 enzyme, by Thermo Fisher Scientific (1 mentions)
Icycler iq pcr plates, by Bio-Rad (1 mentions)
4 protocols using nanodrop 1000
1
Gene Expression Quantification Protocol
2
Isolation of Peripheral Blood Mononuclear Cells for Tisagenlecleucel CAR T-Cell Therapy
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Peripheral blood or bone marrow was collected into EDTA tubes from patients receiving tisagenlecleucel CAR T cell therapy as part of their standard of care for routine clinical testing. Isolation of the mononuclear cells (MNCs) was performed using a density gradient with centrifugation [15 (link)] (STEP 4), and DNA was extracted from the MNCs using the QIAamp DNA Blood Mini Kit as described previously [18 (link),25 (link),26 (link)] (Qiagen, Hilden, Germany). Extracted DNA was quantified using the Nanodrop 1000 (Labtech Ltd., East Sussex, UK) and diluted to 10 ng/µL (STEP 5). When CD3 T cells were isolated, Miltenyi Biotec microbead kits were used.
3
RNA Quality Assessment Workflow
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The concentration and quality of the RNA was assessed spectrophotometrically at 230, 260 and 280 nm (NanoDrop 1000, LabTech, Heathfield, UK). RNA quality and integrity were evaluated by electrophoresis on 1% agarose gel and the RNA integrity number (RIN) was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.).
4
Quantitative PCR Analysis of Tissue Engineered Constructs
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Another set of TE constructs (n = 6 per group) were prepared and either nonimplanted (day 0) or implanted for 7 and 28 days, retrieved, and stored at -80°C. The frozen explanted constructs were then finely chopped with a scalpel blade, and total RNA was extracted by adding 1 mL of TRIzol® (Thermo Scientific). The RNA concentration and purity were determined using a NanoDrop spectrophotometer (NanoDrop 1000; LabTech). cDNA was obtained after reverse transcription of 3 μg of purified RNA using the Superscript II enzyme (Life Technologies) and random primers. Quantitative PCR was then performed using 25 and 75 ng cDNA in 96-well plates (iCycler iQ PCR plates; Bio-Rad) and TaqMan gene expression assays (Life Technologies) for mouse genes and human genes, respectively, following the manufacturer's instructions and using the MyiQ™ Single-Color Real-Time PCR Detection System (Bio-Rad). The results were normalized to those of the respective GAPDH as internal standard. The full names of the genes monitored and the assay IDs are given in Supplementary Table S1.
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