Total RNA from undifferentiated ASCs and from ASCs that had undergone adipogenic differentiation was extracted using RNA extraction kit (Qiagen, Germantown, MD, USA) and then digested with DNase I (Qiagen)). A total of 1 μg of mRNA was used for cDNA synthesis with an Applied Bioscience purification kit (Thermo Fisher Scientific, USA). qRT-PCR was performed using the SYBR Green qPCR SuperMix (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Oligonucleotide primers were designed with the vendor’s software (IDT, USA). Table 2 lists the primer sequences used for qRT-PCR. PCR conditions were: 2 min at 95 °C, and 40 cycles of 15 s at 95 °C and 30 s at 60 °C. The target and reference genes were amplified in separate wells. All reactions were performed in duplicate. The 2−ΔΔCT method was used to quantify gene expressions and data were normalized to GAPDH, which was used as an internal control.
Applied bioscience purification kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Applied Bioscience purification kit is a laboratory equipment product designed for the purification of biological samples. The kit contains the necessary components and reagents to efficiently extract and purify target molecules from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using applied bioscience purification kit
1
Quantitative Analysis of Adipogenic Gene Expression
2
Quantitative RT-PCR of ASC Transcripts
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Total RNA from ASCs was extracted using an RNA extraction kit (Qiagen, Germantown, MD, USA). One microgram of mRNA was used for cDNA synthesis with an Applied Bioscience purification kit (Thermo Fisher Scientific, USA). qRT-PCR was performed using the instructions from the SYBR Green qPCR SuperMix (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Oligonucleotide primers were designed using the vendor’s software (IDT, Coralville, IA, USA). Table 2 lists the primer sequences used for qRT-PCR. PCR conditions: 2 min at 95 °C and 40 cycles of 15 s at 95 °C and 30 s at 60 °C. The target and reference genes were amplified in separate wells. All reactions were performed in duplicate. The 2(−∆∆CT) method was used to quantify gene expressions and normalized data to GAPDH, which was used as an internal control.
3
Quantifying Gene Expression in HUVECs
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Total RNA from HUVECs was extracted using an RNA extraction kit (Qiagen, Germantown, MD, USA). A total of 1µg of mRNA was used for cDNA synthesis with an Applied Bioscience purification kit (Thermo Fisher Scientific, Waltham, MA, USA). qRT-PCR was performed using the SYBR Green qPCR SuperMix (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Oligonucleotide primers were designed with the vendor’s software (IDT, Newark, NJ, USA; https://www.idtdna.com/SciTools , accessed on 12 December 2018). Table 2 lists the primer sequences used for qRT-PCR. PCR conditions were: 2 min at 95 °C and 40 cycles of 15 s at 95 °C and 30 s at 60 °C. The target and reference genes were amplified in separate wells. All reactions were performed in duplicate. The 2−∆∆CT method was used to quantify gene expressions and data were normalized to GAPDH, which was used as an internal control.
4
Quantifying Gene Expression in Stem Cells
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The total RNA from undifferentiated ASCs and from induced-ASCs was extracted using an RNA extraction kit (Qiagen, Germantown, MD, USA) and then digested with DNase I (Qiagen, USA). A total of 1 µg of mRNA was used for the cDNA synthesis Applied Bioscience purification kit (Thermo Fisher Scientific, USA). qRT-PCR was performed using the SYBR Green qPCR SuperMix (Bio-Rad, Hercules, CA, USA), according to the manufacturer’s instructions. Oligonucleotide primers were designed with the vendor’s software (IDT, Coralville, IA, USA). Table 2 lists the primer sequences used for qRT-PCR. The PCR conditions were: 2 min at 95 °C and 40 cycles of 15 s at 95 °C and 30 s at 60 °C. The target and reference genes were amplified in separate wells. All reactions were performed in duplicate. The 2−ΔΔCt method was used to calculate the relative fold change in the gene expression after normalization to GAPDH, which was used as an internal control.
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