For RT² Profiler™ PCR Array 10 000 PKH26+ and 10 000 PKH26− cells (selected 12 days after PKH26 staining) were sorted into 100 μl Buffer RL (Norgen) as described before [13] . RNA was isolated with Single Cell RNA Purification Kit (Norgen) with On-Column DNA Removal (Norgen) followed by reverse transcription PCR using NCode Vilo (Invitrogen). RT² Profiler™ PCR Array (Qiagen) detecting murine CSC-related genes was performed using the vendor's protocol and StepOne Plus PCR (Applied Biosystems) cycler. Genes included in the array are listed in Supplementary Table 2.
Ncode vilo
Manufactured by Thermo Fisher Scientific
Sourced in United States
The NCode Vilo is a laboratory equipment product from Thermo Fisher Scientific. It is designed for the isolation, purification, and analysis of RNA samples. The core function of the NCode Vilo is to facilitate the reverse transcription of RNA to cDNA, which is a crucial step in many molecular biology and gene expression studies.
Automatically generated - may contain errors
Lab products found in correlation
On column dna removal, by Norgen Biotek (2 mentions)
Buffer rl, by Norgen Biotek (2 mentions)
Single cell rna purification kit, by Norgen Biotek (2 mentions)
Steponeplus, by Thermo Fisher Scientific (2 mentions)
Rt profiler pcr array, by Qiagen (1 mentions)
Rt2 profiler pcr array, by Qiagen (1 mentions)
Sybr green jumpstart taq readymix, by Merck Group (1 mentions)
Quantitect sybr green, by Qiagen (1 mentions)
Revertaid premium first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 2, by Roche (1 mentions)
Qiazol, by Qiagen (1 mentions)
3 protocols using ncode vilo
1
Profiling Murine Cancer Stem Cells
2
Quantifying Murine Cancer Stem Cells
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For the RT2 Profiler PCR Array analysis, we sorted 2000–10,000 MIC+ and MIC− B16-F10 cells in 100 μL of Buffer RL (Norgen, Thorold, Canada). Next, RNA was isolated using the Single Cell RNA Purification Kit (Norgen, Thorold, Canada) with the On-Column DNA Removal (Norgen, Thorold, ON, Canada) step according to the vendor’s protocol. RNA was eluted using 10 μL of DNase RNase-free H2O (this step was repeated 4 times to increase the RNA yield). Isolated RNA was entirely used for the RT-PCR using NCode Vilo (Invitrogen, Waltham, MA, USA), and obtained cDNA was diluted 6 times. The RT2 Profiler PCR Array (Qiagen, Germantown, MD, USA) detecting murine CSC-related genes was performed according to the vendor’s protocol using the Applied Biosystems StepOne Plus (Waltham, MA, USA) device. The list of genes is included in Table S1 .
3
RNA extraction and qRT-PCR analysis
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RNA was isolated using QIAzol (Qiagen) reagent according to the manufacturer's instructions and reverse-transcribed into cDNA using a RevertAid Premium First Strand cDNA Synthesis Kit (Fermentas) and nCodeVilo (Invitrogen) for analysis of miRNA. Realtime PCR was performed using QuantiTect SYBR Green (Qiagen) or SYBR Green JumpStart Taq Ready Mix (Sigma) on a Light Cycler 480 II (Roche) or a StepOne Plus (Applied Biosystems) platform. Gene expression was calculated according to delta Ct or delta delta Ct methods with EF2 and U6 as reference genes for mRNA and miRNA analysis, respectively.
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