Epididymal adipose tissues were homogenized using the TissueLyser system (#85300, Qiagen, Venlo, The Netherlands), and total RNA was isolated using TRIzol reagent (#15596018, Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA content and purity were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). One microgram of total RNA was reverse-transcribed into cDNA using the AMPIGENE® cDNA synthesis kit (#END-KIT106, Enzo Life Sciences, Farmingdale, NY, USA) and a SimpliAmp Thermal Cycler (Applied Biosystems, Waltham, MA, USA) according to the manufacturer’s instructions. Real-time PCR was performed using AMPIGENE® qPCR Green Mix Hi-ROX (#ENZ-NUC104, Enzo Life Sciences) on a StepOne Plus real-time PCR system (Applied Biosystems). The thermal cycles were as follows: 94 °C for 3 min, followed by 40 cycles at 95 °C for 10 s, 60 °C for 15 s, and 72 °C for 20 s. Mouse 18s rRNA was used as a reference gene, and relative gene expression levels were analyzed using the 2−ΔΔCt method. The primers used for the real-time PCR analysis are listed in Supplementary Materials Table S1 .
Ampigene qpcr green mix hi rox
Manufactured by Enzo Life Sciences
Sourced in United States
AMPIGENE qPCR Green Mix Hi-Rox is a ready-to-use solution for quantitative real-time PCR. It contains all the necessary components, including a proprietary DNA polymerase, buffer, dNTPs, and a green fluorescent dye for detection.
Automatically generated - may contain errors
Lab products found in correlation
Ampigene cdna synthesis kit, by Enzo Life Sciences (8 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (7 mentions)
Rnaiso plus, by Takara Bio (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Tissuelyser system, by Qiagen (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
All in one rt mastermix, by Applied Biological Materials (2 mentions)
Simpliamp thermal cycler, by Thermo Fisher Scientific (1 mentions)
Abi stepone real time pcr system, by Thermo Fisher Scientific (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Qiazol, by Qiagen (1 mentions)
Oligo dt 15 primer, by Promega (1 mentions)
Purelink dnase set, by Thermo Fisher Scientific (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Easy blue total rna extraction kit, by iNtRON Biotechnology (1 mentions)
Lightcycler nano instrument, by Roche (1 mentions)
Cdna master mix, by Toyobo (1 mentions)
Rnaiso plus reagent, by Takara Bio (1 mentions)
20 protocols using ampigene qpcr green mix hi rox
1
Quantification of Gene Expression in Epididymal Adipose Tissue
2
Isolation and qRT-PCR Analysis of Endothelial Cell-Enriched RNA
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Twenty-four hours after injection of LSS- or OSS-sEVs, mice were euthanized via CO2 inhalation and pressure perfused with saline containing heparin (10 U/mL) via the left ventricle after severing the inferior vena cava. Next, we isolated and carefully cleaned the partially ligated LCA and non-ligated RCA of periadventitial fat, as described previously [1 (link)]. Briefly, we flushed the lumen of both carotid arteries for a few seconds with 200 μL of QIAzol lysis reagent (Qiagen, Hilden, Germany) using a 29-gauge insulin syringe in a microfuge tube. The eluent was used to isolate endothelial cell-enriched RNA with QIAzol (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The concentration of RNA was quantified using a NanoDrop (ND-2000) spectrophotometer. We synthesized single-strand cDNA using M-MLV reverse transcriptase (Promega, Fitchburg, WI, USA) and oligo-dT 15 primer (Promega, Fitchburg, WI, USA). qRT-PCR was performed using AmpiGene qPCR Green Mix Hi-ROX (Enzo Life Sciences, Farmingdale, NY, USA) and gene-specific primers on an ABI StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).
3
Brain Tissue RNA Isolation and qPCR Analysis
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Weighed brain tissue samples were homogenized in ultra-pure-guanidine isothiocyanate (Sigma-Aldrich, Cat# 50,983) using innuSPEED Lysis Tubes P in SpeedMill Plus (Analytik Jena, Germany, Cat# 845-CS-1020250). RNA was isolated using Pure Link RNA mini kit (Invitrogen, Cat# 12183018A) as recommended by the manufacturer. DNA contamination was removed using PureLink™ DNase Set (Invitrogen, Cat# 10,977,035). RNA purity and the concentrations were determined using a NanoDrop spectrophotometer (Epoch, BioTek) and total RNA was converted into cDNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Cat# 4,368,814) according to supplier’s protocol. Quantitative PCR (qPCR) was performed using with the primers shown at Supplementary Table 1. AMPIGENE qPCR Green Mix Hi-ROX (Enzo Life Sciences, Cat# ENZ-NUC104) was used under the following conditions: 95 °C for 2 min for initial denaturation, followed by 5 s (40 cycles) at 95 °C and 30 s at 57 °C. Data were generated in the final step at 95 °C for 15 s and melting curves (65 to 95 °C) were acquired at the end for each primer set. Relative gene expression was calculated by 2−ΔΔCt method by normalizing gene expression to β-actin (Aktas et al. 2015 (link)). Data were generated for four mice per group and each biological replicate has four technical replicates.
4
Real-time PCR Analysis of HO-1 Expression
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Total RNA was extracted using Trizol Reagent (Life Technologies, Carlsbad, CA, USA). One microgram of RNA was reverse-transcribed by an AMPIGENE cDNA Synthesis Kit (Enzo Life Sciences, Inc., NY, USA), and the resultant cDNA was subjected to PCR using AMPIGENE qPCR Green Mix Hi-Rox (Enzo Life Sciences, Inc.). Real-time PCR was performed using the Applied Biosystems StepOne Plus Real-Time PCR System (Applied Biosystems, CA, USA) and PCR conditions for all genes were as follows: 95 ℃ for 2 min, followed by 40 cycles of 95 ℃ for 10 sec and 60 ℃ for 30 sec. The relative amount of each gene was normalized to the amount of GAPDH and calculated using the 2-ΔΔCt method. The qPCR primers were: HO-1 sense, 5′-CCA GCA ACA AAG TGC AAG AAT C-3′, HO-1 anti-sense, 5′-CCA CCA GAA AGC TGA GTG TAA G-3′, GAPDH sense, 5′-GTG GTC TCC TCT GAC TTC AAC-3′, GAPDH anti-sense, 5′-CCT GTT GCT GTA GCC AAA TTC-3′.
5
Pancreatic Tissue RNA Extraction and Expression Analysis
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Total RNA was extracted from the homogenised pancreatic tissue, isolated PACs, and hAT-MSCs using the Easy-BLUE Total RNA Extraction kit (Intron Biotechnology, Seongnam, Korea), according to the manufacturer’s instructions. cDNA was synthesised from 1 μg of total RNA with CellScript All-in-One cDNA Master Mix (CellSafe, Yongin, Korea), and the samples were analysed in triplicate using AMPIGENE qPCR Green Mix Hi-ROX with the SYBR Green dye (Enzo Life Sciences, Farmingdale, NY, USA). The expressions of target genes were analysed according to the 2−ΔΔ/Cts method and normalised to the mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primer sequences used in this study are listed in Additional file 4 : Table S2.
6
Quantification of Gene Expression by qRT-PCR
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Total RNA was extracted using easy-BLUE Total RNA Extraction Kit (INTRON, Daejeon, Korea). The isolated RNA was transcribed by AMPIGENE cDNA Synthesis Kit (Enzo Life sciences, Inc., NY) and real time PCR was performed using the StepOne Real-Time PCR System with AMPIGENE qPCR Green Mix Hi-Rox (Enzo Life sciences, Inc., Farmingdale, NY). Real-time PCR conditions for all genes were as follows: 95°C for 2 min, followed by 40 cycles of 95°C for 10 s and 62°C for 30 s. The relative expression changes of the target genes were quantified by normalizing their expression to that of GAPDH. The PCR primers of all the target genes are listed in Table 1 .
7
Quantitative PCR Analysis of PAIP1 Expression
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1 microgram of RNA was used to reverse transcribe using the AMPIGENE cDNA Synthesis Kit (Enzo Life Sciences, Inc.) according to the manufacturer’s protocol and then, the resultant cDNA was subjected to PCR using the AMPIGENE qPCR Green Mix Hi-Rox (Enzo Life Sciences, Inc.). The real-time PCR was performed using the Applied Biosystems StepOnePlus Real-Time PCR System (Applied Biosystems) and the amplification parameters for all genes were as follows: 95 °C for 2 min, followed by 40 cycles of 95 °C for 10 s, and 60 °C for 30 s. Fold amplification of each gene was normalized to the amount of GAPDH and calculated using the 2−∆∆CT method47 (link). We used the qPCR primers as follows: PAIP1 forward, 5′-TTT GGA AGA TGC TTG GAA GG-3′, PAIP1 reverse, 5′-TGA AGT TGC ATG GAC TCT GC-3′; GAPDH forward, 5′-GTGGTCTCCTCTGACTTCAAC-3′, GAPDH reverse, 5′-CCTGTTGCTGTAGCCAAATTC-3′.
8
RNA Extraction and qPCR Analysis
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Total RNA was extracted with TRI-Solution (Bio Science Technology, Seoul, Korea) according to the manufacturer's instructions. RNA concentration was measured using a multi-plate reader according to the manufacturer's instructions. To quantify gene transcription, cDNA was amplified on a LightCycler Nano Instrument (Roche, Mannheim, Germany) using AmpiGene qPCR Green Mix Hi-ROX (Enzo Life Sciences, Farmingdale, NY, USA) and specific primers. Quantitative real-time PCR is included in Table 1 .
9
Adipose Tissue RNA Extraction and qPCR Analysis
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Adipose tissue was homogenized using the TissueLyser system (85300, Qiagen, Venlo, Netherlands), and total RNA was isolated using the TRIzol reagent (15596018, Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions. The RNA content and purity were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). One microgram of total RNA was reverse-transcribed into cDNA using an AMPIGENE® cDNA synthesis kit (END-KIT106, Enzo Life Sciences, Farmingdale, NY, USA) and a SimpliAmp Thermal Cycler instrument (Applied Biosystems, Waltham, MA, USA). Real-time PCR analysis was performed on an AMPIGENE ® qPCR Green Mix Hi-ROX (ENZ-NUC104, Enzo Life Sciences) using a StepOne Plus real-time PCR system (Applied Biosystems). Mouse 18s rRNA was used as a reference gene, and the relative gene expression levels were analyzed using the 2-∆∆Ct method. The primers used for real-time PCR analysis are listed in Table S2.
10
Quantitative Real-Time PCR Analysis
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Total RNA was isolated using RNAiso Plus (TAKARA, Kyoto, Japan) [22 (link)]. The concentration of the extracted RNA was measured at 260 nm and 280 nm using a spectrophotometer (UL 61010-1, Thermo Fisher Scientific), and only RNA having an A260/A280 ratio of 1.8 to 2.0 was used. cDNA was synthesized using the AMPIGENE® cDNA Synthesis Kit (Enzo Life Sciences, Farmingdale, NY, USA) according to the manufacturer’s protocol. Quantitative real-time PCR analysis was performed using a 20 µL reaction mixture using AMPIGENE® qPCR Green Mix Hi-ROX (Enzo Life Sciences, Farmingdale, NY, USA). The results were calculated using the 2−ΔΔCt analysis method and normalized to the 18s rRNA gene level. Primer sequences for the PCR of the two target genes, KLF15 and REDD1, and the internal reference gene, 18s RNA, are listed in Table 3 .
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