RT-PCR was performed as described previously [9 (link)]. Briefly, 10 ng of total RNA was reverse transcribed (RT) using a Taqman microRNA RT kit (Thermo Fisher Scientific) and specific RT primers. Q-PCR was carried out using Taqman 2X universal PCR master mix (no AmpErase UNG) and 20X MicroRNA Assays (Thermo Fisher Scientific) with specific primer for miR-146a, which is designed to detect and quantify mature miRNAs in real time using a 7300 PCR System (Thermo Fisher Scientific). Each sample was run in triplicate. Signals were normalized to U6 housekeeping miRNA run simultaneously. A comparative threshold cycle (Ct) method (ΔΔCt) was used to calculate relative miRNA expression between treated and corresponding control-transfected samples.
Taqman 2x universal pcr master mix no amperase ung
Manufactured by Thermo Fisher Scientific
Sourced in Canada
Taqman 2X universal PCR master mix (no AmpErase UNG) is a ready-to-use solution for real-time PCR amplification. It contains all the necessary components, including DNA polymerase, dNTPs, and buffer, to perform quantitative PCR reactions. The mix does not include the UNG enzyme, which is used to prevent carryover contamination.
Automatically generated - may contain errors
Lab products found in correlation
7300 pcr system, by Thermo Fisher Scientific (1 mentions)
Taqman microrna rt kit, by Thermo Fisher Scientific (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Taqman probe, by Thermo Fisher Scientific (1 mentions)
Real time pcr stepone plus, by Thermo Fisher Scientific (1 mentions)
Microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Cfx384 thermocycler, by Bio-Rad (1 mentions)
Taqman mirna primers, by Thermo Fisher Scientific (1 mentions)
Taqman mirna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
4 protocols using taqman 2x universal pcr master mix no amperase ung
1
Real-Time PCR for miRNA Quantification
2
Quantitative Analysis of miRNA and mRNA
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Total small RNA was extracted with miRVana miRNA isolation kit (Thermo Fisher), and quality and quantity determined by NanoDrop spectrophotometer. miRNA was assessed by real-time PCR using TaqMan probe (Thermo Fisher) and primer sets using Applied Biosystems Real Time PCR StepOne Plus. Briefly, total small RNA (10 ng) was reverse-transcribed using Taqman MicroRNA Reverse Transcription Kit and amplified using TaqMan 2x Universal PCR Master Mix, No AmpErase UNG (Thermo Fisher). To determine VEGFA, ACTB and EP300 mRNAs, one-step reverse transcription coupled with real-time PCR was performed with 2x VeriQuest Probe One-Step qRT-PCR Master Mix (Affymetrix) using total RNA (0.5 μg) extracted with Trizol in an Applied Biosystems Real Time PCR StepOne Plus machine. RT-qPCR probes for the Taqman Gene Expression Assay, i.e. VEGFA (Hs00900055_m1), ACTB (Hs99999903_m1) and EP300 (Hs00914223_m1) were from Thermo Fisher (Cat. #4331182): The primers for semi-quantitative RT-PCR were: RT_ACTB-f: 5΄-ATGGATGATGATATCGCCGCG-3΄; RT_ACTB-r: 5΄-CTAGAAGCATTTGCGGTGGAC-3΄; RT_VEGFA-f: 5΄-ACAGAACGATCGATACAGAA-3΄; RT_VEGFA-r: 5΄-AAAGATCATGCCAGAGTCTC-3΄.
3
Reverse Transcription and qPCR of Embryo miRNAs
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Reverse transcription of whole embryo lysate samples was performed using a MicroRNA Reverse Transcription kit (Thermo Fisher Scientific, P/N: 4366596) in addition to separately ordered TaqMan miRNA primers (Thermo Fisher Scientific, P/N: 4427975) for each of the 6 targeted miRNAs and snU6 according to the manufacturer’s instructions: 10 µL of RT Mastermix, 1 µL of whole embryo lysate and 4 µL of water were added to each RT reaction before thermal cycling at 16 °C for 30 min, 42 °C for 30 min and 85 °C for 5 min in both cases. For qPCR amplification of whole embryo lysate samples, 1.33 µL of RT reaction products was mixed with 18.67 µL of TaqMan 2X Universal PCR Mastermix No AmpErase UNG (Thermo Fisher Scientific, P/N: 4324018), and quantitative amplification was performed using a CFX384 thermocycler (BioRad, Mississauga, ON, Canada) for 40 cycles.
4
miRNA Profiling in Enteroids
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For miRNA profiling, the total small RNA was extracted from enteroids treated with FCMs of B6, db/db, and DIO, using miRNeasy Mini kit (Qiagen) and analysed by the NanoString nCounter miRNA Assays, using 50–100 ng of total RNA from each sample in four replicates, and using eight positive control probes and eight negative control probes. Data were analysed using nSolver analysis software V.4.0. Each miRNA background correction count was carried out by subtracting mean+2 SD of eight negative control probes as a cut-off. The miRNA of count 50 or more after background correction was further used for analysis. The individual miRNA expression was quantified, using qRT-PCR by converting small RNAs to cDNA using TaqMan miRNA Reverse Transcription kit (ThermoFisher) and TaqMan 2X Universal PCR Master Mix, No AmpErase UNG (ThermoFisher) on 7900 real-time PCR machine. The TaqMan primers were used for specific miRNAs, and RNU6B was used as an internal control as mentioned in online supplemental table 11 ; and relative expression was calculated using ΔΔCT method.
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