The miRNeasy isolation kit and the QuantiNova SYBR Green PCR mix were purchased from Qiagen (Valencia, CA). Running buffer, transfer buffer, NuPage 4–12% Bis-Tris gels, and Invitrolon polyvinylidene fluoride (PVDF) membranes (0.45 µm) were procured from Invitrogen (Carlsbad, CA) while the blocking buffer was from Li-Cor Biosciences (Lincoln, NE). Antigen retrieval solution and 4',6-diamidino-2-phenylindole (DAPI) were from Vector Laboratories (Burlingame, CA), and the normal donkey serum was purchased from Jackson ImmunoResearch Laboratories (Westgrove, PA). The following primary antibodies were used for immunohistochemistry and western blotting: rabbit monoclonal antibody against aquaporin 5 (1:100 or 1:5,000; Abcam Inc., Cambridge, MA); mouse monoclonal antibody against E-cadherin (1:100 or 1:5,000; BD Biosciences, San Jose, CA), rabbit polyclonal antibody against α-SMA (1:100 or 1:5,000; Abcam Inc.), and mouse monoclonal antibody against beta actin (1:10,000; Sigma-Aldrich Co., St. Louis, MO). Fluorescein isothiocyanate (FITC) or tetramethylrhodamine (TRITC) conjugated secondary antibodies (1:100, Jackson ImmunoResearch Laboratories) were used for immunohistochemical staining, while IRDye 680RD or IRDye 800CW secondary antibodies (1:5,000; Li-Cor Biosciences) were used for western blotting.
Quantinova sybr green pcr mix
Manufactured by Qiagen
Sourced in Germany
The QuantiNova SYBR Green PCR mix is a ready-to-use solution for quantitative real-time PCR (qPCR) experiments. It contains all the necessary reagents, including the SYBR Green I dye, for the detection and quantification of DNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Irdye 800cw secondary antibody, by LI COR (1 mentions)
Mirneasy isolation kit, by Qiagen (1 mentions)
Normal donkey serum (nds), by Jackson ImmunoResearch (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Antigen retrieval solution, by Vector Laboratories (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal antibody against beta actin, by Merck Group (1 mentions)
Mouse monoclonal antibody against e cadherin, by BD (1 mentions)
Rabbit polyclonal antibody against α sma, by Abcam (1 mentions)
Irdye 680rd, by LI COR (1 mentions)
Steponeplus, by Thermo Fisher Scientific (1 mentions)
Quantitect primer assay, by Qiagen (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Rotor gene q real time pcr system, by Qiagen (1 mentions)
Omniscript rt kit, by Qiagen (1 mentions)
3 protocols using quantinova sybr green pcr mix
1
Quantification of Protein Expression in Cells
2
Quantitative analysis of RNA expression
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Total RNA was extracted from parental and spheroid cells from MCF-7 and MDA-MB-453 using the miRNeasy Mini kit (Qiagen, Germany) following the manufacturer's instructions. Reverse transcription was carried out using the Quantitect Reverse Transcription kit (Qiagen, Germany) using 1μg RNA. The cDNA levels were quantified by Applied Biosystems StepOne Plus (Applied Biosystems, USA) using the SYBR green assay (Quantinova SYBR green PCR mix, Qiagen Germany). Pre-designed primers (Quantitect Primer Assay) specific to the genes of interest were obtained from Qiagen, Germany. The qPCR results were analyzed using StepOne™ Software v2.3. GAPDH has been used as the endogenous control.
3
Semi-quantitative RT-PCR Analysis of p53 Isoforms
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Semi-quantitative RT-PCR analysis was performed with Qiagen RotorgeneQ Real-time PCR system using SYBR-Green detection according to manufacturer’s instructions. Briefly, total RNA was extracted from BMMCs using the Trizol®/Qiagen RNeasy hybrid RNA extraction kit (Qiagen, Germany) and the RNase-free DNase-treatment kit (Qiagen, Germany). 1μg of the obtained DNA-free RNA was reverse-transcribed using Omniscript® RT Kit (Qiagen, Germany). Each PCR assay was performed in a 20uL reaction containing QuantiNova SYBR Green PCR mix (Qiagen, Germany), primers specific for each transcript (Additional file 1 ) and 3uL of 5-fold diluted cDNA (equivalent to 30 ng). From a panel of 10 reference genes, RPL27, CYC1 and RNU6–1 were selected as the most stable using geNorm, Normfinder and BestKeeper. The sequences and location of all primers are presented in Additional file 1 . Each gene analysis was performed in triplicate and real-time PCR products were verified by melting curve analysis. Cq (threshold cycle) was determined for each assay (software) and Cq value > 35 was interpreted as non-expressed. The relative expression of p53 isoform transcripts was normalized to a geometric average expression level of the 3 reference genes and expressed as the fold change to calibrator (healthy marrow) calculated using Delta-Delta Cq method.
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