Keratinocytes were grown to confluence in 6-well tissue culture plates. For comparison between cell lines (Figure S1D ), medium was switched from K-SFM to differentiation medium 24 hours before sample collection. For scratch wound experiments (Figure 2C ), culture medium was switched from K-SFM to differentiation medium, then treatment samples were scratched with a 1000 mL pipette tip in a cross-hatch pattern. The scratch procedure was repeated every other day for 10 days. Samples were collected one day following the last scratch procedure. RNA was isolated from the samples using RNeasy spin column kits (Qiagen #74004) and reverse transcription was performed using SuperScript IV First-Strand Synthesis System with oligo(dT)20 primers (Invitrogen #18091050). Real-time PCR using a C1000 thermal cycler with a CFX96 optical reaction module (Bio-Rad), iTaq Universal SYBER Green Supermix (Bio-Rad), and transcript-specific primers (Table S1 ) was performed to quantify transcript levels. The 2−ΔΔCT method12 was used to compare each individual keratin to the mean level of all keratins measured.
Rneasy spin column kit
Manufactured by Qiagen
Sourced in Germany, Japan
The RNeasy spin column kit is a lab equipment product designed for the rapid and efficient purification of high-quality RNA from a variety of sample types. The core function of the kit is to utilize a silica-based membrane technology to selectively bind and isolate RNA molecules, while effectively removing contaminants and impurities.
Automatically generated - may contain errors
Lab products found in correlation
Thermal cycler dice, by Takara Bio (3 mentions)
Primescript rt master mix, by Takara Bio (3 mentions)
Isogen, by Nippon Gene (3 mentions)
Taqman assay, by Thermo Fisher Scientific (3 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (3 mentions)
Superscript 3, by Thermo Fisher Scientific (3 mentions)
Dnase 1, by Qiagen (3 mentions)
Tb green premix ex taq 2, by Takara Bio (2 mentions)
Superscript 2 first strand synthesis kit, by Thermo Fisher Scientific (2 mentions)
Easysep mouse epithelial cell enrichment kit, by STEMCELL (2 mentions)
Anti α6 integrin cd49f, by BD (2 mentions)
Epcam fitc conjugated antibody, by BioLegend (2 mentions)
Dneasy blood and tissue spin column kit, by Qiagen (2 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (2 mentions)
Facsaria, by BD (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Cfx96 optical reaction module, by Bio-Rad (1 mentions)
Itaq universal syber green supermix, by Bio-Rad (1 mentions)
Superscript 4 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Oligo dt 20 primer, by Thermo Fisher Scientific (1 mentions)
23 protocols using rneasy spin column kit
1
Quantifying Keratin Expression in Keratinocytes
2
Quantitative RT-PCR Gene Expression Analysis
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Total RNA was extracted with Isogen (Nippon gene, Japan) and RNeasy spin column kits (Qiagen, USA). First-strand cDNA was synthesized from total RNA using PrimeScript RT Master Mix (Takara Bio, Inc.) and subjected to real time RT-PCR using TB Green Premix Ex Taq II (Takara Bio, Inc.) with Thermal Cycler Dice (Takara Bio, Inc.) according to the manufacturer’s instructions. Gene expression levels were normalized relative to those of the housekeeping gene Rplp0. Primer sequences for each gene are listed in Additional file 1 : Table S1.
3
RNA Extraction and RT-qPCR Analysis
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Total RNA was extracted with Isogen (Nippon Gene) and RNeasy spin column kits (Qiagen). First-strand cDNA was synthesized from the total RNA using PrimeScript RT Master Mix (Takara Bio) and subjected to real-time RT-PCR using TB Green Premix Ex Taq II (Takara Bio) with Thermal Cycler Dice (Takara Bio) according to the manufacturer’s instructions. Gene expression levels were normalized relative to those of the housekeeping gene RPLP0 (Rplp0). Primer sequences for each gene are listed in Supplemental Table 4 .
4
Enrichment and Sorting of Mammary Tumor Cells
5
Enrichment and Sorting of Mammary Tumor Cells
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Mammary tumors were dissociated into single cell suspensions through mechanical separation and enzymatic digestion as described22 (link). Dissociated tumor cells were enriched for Lin− (CD45−/ CD31−/ TER119−/ BP-1−) mammary epithelial cells with StemCell Technologies EasySep Mouse Epithelial Cell Enrichment Kits per the manufacturer’s instructions. Lin− cells were then incubated on ice for 20 min with anti-CD49f (α6 integrin) (BD Biosciences 555734) together with Alexafluor 647 (Invitrogen A21247) in PBS. Cells were spun down for 5 min at 550x g, then incubated with EpCAM-FITC conjugated antibody (Biolegend 118208) in PBS. Tumor cells were sorted on a BD FACS Aria cell sorter machine equipped with Diva software into their luminal (Lin−/ CD49flow/EpCAMhigh) and basal (Lin−/CD49fhigh/EpCAMlow) subpopulations. Sorted cells were collected into 15ml conical tubes containing PBS. Genomic DNA was collected from sorted cell populations using Qiagen Blood and Tissue DNeasy spin column kit. Total RNA was collected from sorted cell populations using Qiagen RNeasy spin column kit. RNA was reversed transcribed using Invitrogen Superscript II First Strand Synthesis kit.
6
Quantifying Gene Expression in Mouse Lung and Calu-3 Cells
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RNA was isolated from whole mouse lung tissue or Calu-3 cells using the RNeasy Spin column kit (Qiagen #75144) then reverse transcribed using the cDNA Reverse Transcription Kit (Applied Biosystems #4308228) as previously described46 (link). Gene expression was reported as fold change over WT-saline condition normalized to GAPDH in mouse lung tissues or OAZ1 for Calu-3 cells and calculated using the double delta CT (2∆∆CT) method as previously described7 (link),8 (link),46 (link).
7
Circadian Analysis of ELGs in Mice
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After dietary intervention, ELGs were collected from euthanized mice every 3 hours for eight time points over the circadian cycle (ZT0, ZT3, ZT6, ZT9, ZT12, ZT15, ZT18, and ZT21) at 12 weeks for the NC-1 and HFD groups, as previously described.58 (link),63 (link) Briefly, total RNA was isolated from the ELGs of NC-1 and HFD animals collected at the different ZTs using the RNeasy Spin Column Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. At each time point, RNA-Seq analysis was performed using three biological replicates. In addition, tissue samples that were collected during the dark phase, when the animals were kept in the dark, were collected under dim red illumination (3-watt safe lamp, DG-20A; Jining Hengshuo Testing Instrument Co., Ltd., Shandong, China), as previously described.64 (link)
8
Quantitative RT-PCR for Gene Expression
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Quantitative reverse transcriptase PCR was performed and analyzed as before43 . Cells were scraped off plates and then lysed in lysis buffer per manufacturer’s protocol. RNA was purified using an RNeasy spin column kit (QIAGEN). We converted RNA by reverse transcription and then cDNA was quantified by real time PCR, using HPRT, as a control to assess target gene regulation.
9
Quantifying Transcripts in Cells
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RNA was extracted using the RNeasy spin column kit (Qiagen), plus DNase treatment to eliminate gDNA. cDNA was generated with SuperScript III (Invitrogen), and quantitative RT-PCR was performed using Taqman Universal PCR Master Mix (Applied Biosystems). The following Taqman assays (Life Technologies) were used: mOlig2 (Mm01210556_m1), mGAPDH (Mm99999915_g1), hTP53 (Hs01034249_m1) and hGAPDH (Hs02758991_g1).
10
RNA Extraction from Adherent Cells and Spheroids
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RNA was extracted using the RNEasy Spin Column kit (cat. 74104; Qiagen) according to the manufacturer’s protocol, with the optional DNaseI (cat. 79254; Qiagen) treatment. Adherent cells were collected by aspirating medium and scraping cells into 600 μL Buffer RLT and stored at − 80 °C until processing. Spheroids were pelleted at 800 g, 4 °C, medium was aspirated, and cells were lysed in 350 μL Buffer RLT and stored at − 80 °C until processing. RNA concentration, A260/280, and A280/230 were determined using a NanoDrop One Microvolume UV–Vis Spectrophotometer (Thermo Scientific).
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