RNA from EXOs was isolated using miRNeasy columns (Qiagen) according to the manufacturer’s protocol; total RNA from cell lines was extracted with a TRIzol extraction kit (Life Technologies, Grand Island, NY, USA). Nucleic acid quality and quantity were assessed with a NanoDrop spectrophotometer (NanoDrop Technologies, ThermoFisher Scientific). Total RNA for each sample was reverse transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit (Life Technologies) according to the manufacturer’s protocols. qPCR was performed on a ViiA7 system (Life Technologies) using TaqMan PCR Master Mix (Life Technologies). Predesigned TaqMan probes (Life Technologies) were used for Matrix Metalloproteinase 1 (MMP1, Hs00899658_m1), Matrix Metalloproteinase 9 (MMP9, Hs00957562_m1), Cyclin D1 (CCND1, Hs00765533_m1), CD99 (Hs00908458) and miR-199a-3p (Hs002304). Relative quantification as performed with the ΔΔCT method, and the expression levels of the target genes were normalized to those of the housekeeping gene GAPDH (Hs99999905_m1), RNU6b (Hs001093) or miR-16 (Hs000391).
Taqman predesigned probes
Manufactured by Thermo Fisher Scientific
Sourced in United States
TaqMan predesigned probes are DNA or RNA detection reagents designed for use in quantitative PCR (qPCR) assays. They contain a fluorescent reporter dye and a quencher dye, which allows for the detection and quantification of target nucleic acid sequences.
Automatically generated - may contain errors
Lab products found in correlation
Taqman pcr master mix, by Thermo Fisher Scientific (2 mentions)
Viia 7 system, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Steponeplus, by Thermo Fisher Scientific (2 mentions)
Trizol extraction kit, by Thermo Fisher Scientific (1 mentions)
Mirneasy column, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rnu6b, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Taqman gtxpress master mix, by Thermo Fisher Scientific (1 mentions)
Illustratmblood genomicprep mini spin kit, by GE Healthcare (1 mentions)
5 protocols using taqman predesigned probes
1
Quantitative Analysis of Exosomal RNA
2
Quantitative Analysis of miR-214-3p and HMGA1 Expression
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RNA from cell lines and tissues was extracted by using the TRIzol reagent following the manufacturer’s instructions (Life Technologies, Grand Island, NY, USA) and nucleic acid quality and quantity were assessed by using a NanoDrop spectrophotometer (NanoDrop Technologies LLC, Wilmington, DE, USA). The total RNA from each sample was reverse-transcribed into cDNA by using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocols. qRT-PCR was performed by using a ViiA7 system (Life Technologies) and TaqMan PCR Master Mix (Life Technologies). Predesigned TaqMan probes (Life Technologies) were used for miR-214-3p (assay ID: 002306) and for HMGA1 (assay ID: Hs00431242_m1).
Relative quantification was performed by using the ΔCT method, the expression levels of the target genes were normalized to those of the housekeeping gene glycer-aldehyde-3-phospate dehydrogenase (GAPDH) (assay ID: Hs99999905_m1), or RNU6B (assay ID: 001093) (Life Technologies).
Relative quantification was performed by using the ΔCT method, the expression levels of the target genes were normalized to those of the housekeeping gene glycer-aldehyde-3-phospate dehydrogenase (GAPDH) (assay ID: Hs99999905_m1), or RNU6B (assay ID: 001093) (Life Technologies).
3
Quantifying Gene Expression Profiles in Angiogenic Pathways
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The relative gene expression quantification was performed using a ‘TaqMan Gene Expression Master Mix’ (10X) (Thermo Fisher Scientific Inc.) in a final reaction volume of 10 µl in a Step One Plus (Thermo Fisher Scientific Inc.) real-time PCR detection machine. TaqMan predesigned probes (Thermo Fisher Scientific Inc.) for the target genes VEGFA, VEGFR2, IL8, Ki-67, uPar, FVIII, VEGFC, VEGFR3, LYVE-1, and PDPN were used for this study (Supplementary Table S1, Supplemental digital content 1, http://links.lww.com/MR/A333 ). Expression data were calculated using 2−ΔΔCt [29 (link)]. The reference genes used to normalize the variations between samples were hypoxanthine phosphoribosyltransferase 1 and b-actin (ACTB) (García-P, et al. 2021) [30 (link)]. Every sample was run in triplicate. A nontemplate control was included in every reaction. A control sample was used as an internal calibrator and run in every plate to normalize for interplate variation.
4
Quantitative Gene Expression Analysis
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The relative gene expression quantification was performed using a “TaqMan Gene Expression Master Mix” (10×) (Applied Biosystems, Foster City, CA, USA) in a final reaction volume of 10 µL in a Step One Plus (Applied Biosystems, Foster City, CA, USA) real-time PCR detection machine. TaqMan predesigned probes (Thermo Fisher Scientific Inc., Waltham, MA, USA) for the four housekeeping ACTB/TFRC/HPRT1/TBP genes that were used for this study (Table 2 ). Every sample was run in triplicate. A non-template control was included in every reaction. A control sample was used as an internal calibrator and run in every plate to normalize for inter-plate variation.
5
UGT2B7 Genotyping from Blood Samples
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Concerning DNA extraction and genotyping of UGT2B7 selected SNPs, 5 mL blood samples were collected from each participant in EDTA vacutainers before drug administration. The DNA extraction was performed using the illustraTM blood genomicPrep Mini Spin Kit (GE Healthcare UK Limited, Amersham, UK). The concentration of the extracted genomic DNA was measured using the NanoDrop™ ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The gDNA isolates were all used undiluted in the SNP analysis.
Genotyping of UGT2B7 rs7438135 and rs11740316 was performed using TaqMan™ predesigned probes (Thermo Fisher Scientific, Waltham, MA, USA) and the Rotor-Gene QTM real-time PCR instrument (QIAGEN, Hilden, Germany). The reaction plate was prepared using TaqMan™ GTXpress™ Master Mix (Thermo Fisher Scientific, Walthman, MA, USA), gDNA, and RNAse-free water. The thermal profile was as follows: denaturation of the DNA strand at 95 °C for 20 s, hybridization of the primers and probes at 92 °C for 40 s, then elongation at 60 °C for 30 s.
Genotyping of UGT2B7 rs7438135 and rs11740316 was performed using TaqMan™ predesigned probes (Thermo Fisher Scientific, Waltham, MA, USA) and the Rotor-Gene QTM real-time PCR instrument (QIAGEN, Hilden, Germany). The reaction plate was prepared using TaqMan™ GTXpress™ Master Mix (Thermo Fisher Scientific, Walthman, MA, USA), gDNA, and RNAse-free water. The thermal profile was as follows: denaturation of the DNA strand at 95 °C for 20 s, hybridization of the primers and probes at 92 °C for 40 s, then elongation at 60 °C for 30 s.
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