Cells were lysed using RealTime ready cell lysis buffer (Roche Life Science, 7248431001) with 1:80 diluted RNAse inhibitor (Sigma, 3335402001) and DNAseI (Thermo Fisher Scientific, AM2222). 96‐well plates were lysed by aspirating the media the cells and adding 50 μl lysis buffer per well. The plates were shaken at 1,250 RPM for 25 min. The lysate was mixed with a pipette 10 times and checked for lysis under the microscope. Plates were frozen for at least 15 min at −80°C. Lysates were thawed on ice and diluted 1:5 with nuclease‐free water. 2.25 μl of diluted total lysate was used in a PreAmp reaction with the FMR1‐FAM and POP4‐ABY TaqMan assays (5 μl PreAmp Master Mix (Applied Biosystems 4488593), 2.5 μl 0.2× diluted FMR1 and POP4 assays, 0.25 μl 40× TaqMan RT enzyme mix (Applied Biosystems A36107C), and 2.25 μl lysate). The preamp reaction was run for 12 cycles in an Applied Biosystems QS7 machine. PreAmp reactions were diluted 1:5 and 2 μl used in a qPCR reaction with the same two TaqMan assays (5 μl TaqMan Fast Advanced Master Mix (Applied Biosystems 4444557), 0.5 μl 20× FMR1‐FAM TaqMan assay, 0.5 μl 20× POP4‐ABY TaqMan assay, 2 μl Nuclease‐Free water) and run on a QuantStudio 7 (Thermo Fisher Scientific). Specific probe information is given in Table 2 .
Realtime ready cell lysis buffer
Manufactured by Roche
The RealTime Ready Cell Lysis Buffer is a laboratory reagent designed to facilitate the lysis (breakdown) of cells. It is a component typically used in molecular biology and genomics workflows to prepare samples for downstream analysis, such as gene expression studies or nucleic acid extraction.
Automatically generated - may contain errors
Lab products found in correlation
Np 40, by Thermo Fisher Scientific (3 mentions)
Thermomixer c, by Eppendorf (2 mentions)
Rnase free water, by Takara Bio (2 mentions)
Rnasein plus, by Promega (2 mentions)
Poly d lysine, by Merck Group (2 mentions)
Gelatin, by Merck Group (2 mentions)
Collagen 1, by BD (2 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Preamp master mix, by Thermo Fisher Scientific (1 mentions)
Taqman fast advanced master mix, by Thermo Fisher Scientific (1 mentions)
Rnase inhibitor, by Merck Group (1 mentions)
Quantstudio 7, by Thermo Fisher Scientific (1 mentions)
Qs7 machine, by Thermo Fisher Scientific (1 mentions)
Xf glycolysis stress test kit, by Agilent Technologies (1 mentions)
Reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (1 mentions)
Tryple select enzyme, by Thermo Fisher Scientific (1 mentions)
Nextseq 500 system, by Illumina (1 mentions)
Rnase free water, by Thermo Fisher Scientific (1 mentions)
7 protocols using realtime ready cell lysis buffer
1
Cell Lysis and Gene Expression Quantification
2
Single-Cell RNA Lysis and Preservation
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Total RNAs or sorted single cells were diluted or lysed in 1 μL of cell lysis buffer containing 1 U RNasein plus (Promega), 10% RealTime ready Cell Lysis Buffer (Roche), 0.3% NP40 (Thermo Fisher), and RNase-free water (TaKaRa). The lysate solution was immediately centrifuged and mixed using a ThermoMixer C (Eppendorf) at 2000 rpm for 1 min at 4 °C. The cell lysate solution was stored at −80 °C until use.
3
Glycolytic Flux Measurement in Cell Types
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Glycolysis in the same number of live cells was measured using the XF Glycolysis Stress Test kit according to the manufacturer's instructions (Agilent). The glycolysis kit directly measures extracellular acidification rate (ECAR) and evaluates the glycolytic flux. In brief, cells originating from the muscle or liver were seeded on wells of XF96 microplates coated with collagen1 (BD Biosciences). Cells originating from the lungs were seeded on wells coated with poly-d -lysine (Sigma-Aldrich), and cells originating from the lymph nodes were plated on wells coated with gelatin (Sigma-Aldrich). Cells were plated at 3 × 104 cells/well 24 to 48 hours prior to the assay. Total RNA was extracted using the RealTime Ready Cell Lysis Buffer (Roche) and subjected to qPCR. ECAR values were normalized to Gapdh mRNA in each sample.
4
Mitochondrial Respiration Profiling
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The oxidative phosphorylation kit measures key parameters of mitochondrial function by directly measuring the oxygen consumption rate (OCR). In brief, the same number of cells originating from the muscle or liver was seeded on collagen 1 (BD Biosciences)–coated wells of XF96 microplates. The same number of cells originating from the lung was seeded on poly-d -lysine (Sigma)–coated wells, and cells originating from the lymph were plated on gelatin (Sigma)-coated wells. Cells were plated at 30,000 cells/well 24 to 48 hours prior to the assay. Total RNA was extracted using “RealTime Ready Cell Lysis Buffer” (ROCHE) and subjected to qPCR. ECAR and OCR values were normalized to Gapdh mRNA for each sample. Each data point represents mean ±SD (n > 4).
5
Quantitative RT-PCR Analysis of mRNA
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Extracted RNA was quantified and its quality was assessed by measuring the 260 nm/280 nm ratio. For the mRNA measurements following TRIzol (Invitrogen) isolation, cDNA was first produced using 500 ng RNA and cDNA SuperMix (QuantaBio) and then subjected to qRT-PCR using Blue SYBR low Rox (PCR Biosystems) and qRT-PCR primers. For the mRNA measurements following extraction using the RealTime Ready Cell Lysis Buffer (Roche), RNA was directly subjected to one-step qRT-PCR and reverse transcriptase (Invitrogen) was added to the SYBR mix. mRNA levels were normalized to endogenous Hprt, Rplp0, or Gapdh. The qRT-PCR primers used are listed in Supplementary Table S5.
6
Isolation of Mouse Cumulus-Oocyte Complexes
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Mice were injected for hyperovulation with equine chorionic gonadotropin (eCG, ASKA Pharmaceutical) or CARD HyperOva (KYUDO). Ovaries were collected 48 h after injection and put into M2 medium containing 200 nM 3‐isobutyl‐1‐methyl‐xanthine (IBMX, Sigma) at 37°C. Fully grown follicles were dissected to obtain cumulus–oocyte complexes. From each follicle, an oocyte and a pool of its surrounding cumulus cells were isolated with gentle pipetting. Oocytes at the GV stage and their surrounding cumulus cells were retained for further experiments. After washed in 0.1% PBA in PBS, individual oocytes and their surrounding cumulus cells were manually collected in 10 μl cell lysis buffer [10 U RNasein plus (Promega), 10% RealTime ready Cell Lysis Buffer (Roche), 0.3% NP40 (Thermo Fisher Scientific), and RNase‐free water (TAKARA)] in a 0.2 mL single tube. The collected cell samples were lysed and stored at −80℃ until downstream preparation.
7
Transcriptomic Profile of hiSG Organoids
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On day 80, hiSG organoids were dissociated with TrypLE Select Enzyme (Thermo Fisher Scientific) at 37 °C for 30 min. Live propidium iodide-negative single cells were purified with a FACSAria II system (BD Biosciences) and subjected to droplet-based scRNA-seq on the 10X Genomics Chromium Single Cell 3′ platform (version 2) according to the manufacturer's protocol. Library sequencing was performed via paired-end sequencing with a single sample index on an Illumina NextSeq 500 system. Data preprocessing was carried out with Cell Ranger version 1.2. The subsequent analysis was performed using Seurat (version 4.0.3) and Monocle 3 (version 0.2.2). For the RamDA-seq experiments, sorted cells were collected in 1 μl cell lysis buffer in a 96-well PCR plate (Nippon Genetics). Total RNA was diluted or lysed in 1 μl cell lysis buffer containing 1 U RNasin plus (Promega), 10% Real-Time ready Cell Lysis Buffer (Roche Life Science), 0.3% NP-40 (Thermo Fisher Scientific) and RNase-Free Water (Thermo Fisher Scientific). The lysate solution was immediately centrifuged and mixed using a ThermoMixer C (Eppendorf) for 1 min at 4 °C. The cell lysate solution was stored at -80 °C until use. RNA and sequencing library preparation was performed according to a previous study 36 (link) .
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