For each biological replicate, one whole snap-frozen IBAT pad was pulverized while frozen and mRNA was isolated using the DNeasy blood and tissue spin column kit (Qiagen). cDNA was prepared from 500 ng of mRNA using the qScript cDNA synthesis DNA kit. Perfecta SYBR Green Fastmix (Quanta Biosciences) was used to perform quantitative real-time PCR (MyIQ iCycler real-time PCR thermal cycler, Bio-Rad). See Table 1 for primer sequences. For analysis, the comparative delta Ct method was used as described previously [34 (link)]. The Ct value for the reference transcript was subtracted from the Ct value of the target transcript, providing a delta Ct value. The fold-change between target and reference was determined using the following equation: Fold Changeref = 2-(delta Ct). This manipulation normalized the expression values, allowing us to scale the data with accurate error bars and perform statistical comparisons between conditions. To calculate fold-change between conditions and genotypes, each Fold Changeref value was divided by the mean Fold Changeref of the control condition to obtain a measure of relative expression.
Dneasy blood and tissue spin column kit
Manufactured by Qiagen
The DNeasy Blood and Tissue spin column kit is a DNA extraction and purification system. It is designed to efficiently isolate high-quality genomic DNA from a variety of sample types, including blood, tissue, and cultured cells.
Automatically generated - may contain errors
Lab products found in correlation
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)
Rneasy spin column kit, by Qiagen (2 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (2 mentions)
Facsaria, by BD (2 mentions)
Perfecta sybr green fastmix, by Quanta Biosciences (1 mentions)
5 protocols using dneasy blood and tissue spin column kit
1
Quantitative Real-Time PCR Analysis
2
Molecular Detection of Paenibacillus larvae
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DNA from four dead larvae (stored at −20°C until use) was extracted using a DNeasy Blood and Tissue spin column kit according to manufacturer’s instructions (Qiagen, Valencia CA). PCR was prepared to a 25 µl reaction volume containing 0.5 µM of each forward and reverse primers, 12.5 µl of 2xMidasMix with Taq DNA Polymerase (Monserate Biotechnology Group, San Diego, CA). The MidasMix contained 2 mM MgCl2 and 0.22 mM dNTP; 50–100 ng of DNA template and enough nuclease-free water were added to reach 25 µl.
The primers were designed to amplify a 700-bp region of the P. larvae 16S rRNA gene (Piccini et al. 2002 ). The sequences of forward and reverse primers were as follows:
The primers were designed to amplify a 700-bp region of the P. larvae 16S rRNA gene (Piccini et al. 2002 ). The sequences of forward and reverse primers were as follows:
Forward primer (Pl5): 5’-CGAGCGGACCTTGTGTTTCC-3’
Reverse primer (Pl4): 5’-TCAGTTATAGGCCAGAAAGC-3’
3
Telomere Length Measurement by qPCR
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DNA from whole blood samples was extracted with the DNeasy Blood and Tissue spin column kit (QIAGEN) and telomere length was measured by qPCR as previously described48 (link)–50 ,57 (link). The repeatability of the assay (see Supplementary File 2 for how repeatability was calculated) was 80% and therefore delivers interpretable results65 (link). A full description of our DNA extraction and qPCR protocols including quality control steps can also be found in Supplementary File 2 .
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.
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