RNA was extracted from sorted liver macrophages and from tissue culture by using Tri-Reagent (Sigma-Aldrich) and RNA isolation mini kit (Bioline, Alexandria, Australia) according to the manufacturer’s instructions. RNA was stored in RNase-free water at −80°C. The QuantiTect Reverse Transcription Kit (Qiagen, Melbourne, VIC, Australia) was used to reverse-transcribe up to 1,000 ng total RNA. cDNA (2 µL) was amplified by Quantitative real-time PCR on the Rotor-Gene Q qPCR instrument (Qiagen) with 10 µl reactions using the SensiFAST™ SYBR No-Rox Kit (Bioline). The appropriate oligonucleotide primers were listed in Table 2 , and the reaction was performed under the following conditions: samples were heated at 95°C for 3 min and amplified with 40 cycles of 95°C for 10 s, 60°C for 15 s, and 72°C for 30 s. Reactions were performed in duplicate and gene expression levels were normalized to β-actin. Relative gene expression between samples was calculated using the 2−ΔΔCt calculation method.
Rotor gene q qpcr instrument
Manufactured by Qiagen
The Rotor-Gene Q qPCR instrument is a real-time PCR cycler designed for quantitative PCR analysis. It features a thermal cycler and detection system for analyzing nucleic acid samples.
Automatically generated - may contain errors
Lab products found in correlation
Sensifast sybr no rox kit, by Meridian Bioscience (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Glomelt thermal shift protein stability kit, by Biotium (1 mentions)
3 protocols using rotor gene q qpcr instrument
1
Quantitative real-time PCR analysis of liver macrophages
2
Quantification of miR-200c expression in U2OS cells
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U2OS cells were seeded 24 h prior
to transfection to reach logarithmic growth phase. On the day of transfection,
cells were incubated with polyplexes prepared with either miR-200c
or miR-NC at various N/P ratios in serum-free medium. After 4 h incubation,
polyplexes were completely removed and replaced with culture medium
with 10% FBS for 48 h prior to RT-PCR analysis. Total RNA was extracted
from the transfected U2OS cells using mirVana miRNA isolation kit
(Ambion, USA) according to the manufacturer’s protocol. The
quantification of mature miR-200c using TaqManMicroRNA assay included
a two-step RT-PCR. First, 10 ng of RNA from each sample was converted
to cDNA using TaqMan microRNA reverse transcription cDNA synthesis
kit (Applied Biosystems, California) with specific primers for miR-200c
or internal reference Z30 (Applied Biosystems, California) according
to the manufacturer’s protocol. Second, the PCR amplification
of target cDNA was performed on Rotor-Gene Q qPCR instrument (QIAGEN)
with specific primers for miR-200c or Z30 using AmpliTaq Gold enzymes.
The expression of miR-200c level was normalized to internal miRNA
reference Z30 using comparative CT method.
to transfection to reach logarithmic growth phase. On the day of transfection,
cells were incubated with polyplexes prepared with either miR-200c
or miR-NC at various N/P ratios in serum-free medium. After 4 h incubation,
polyplexes were completely removed and replaced with culture medium
with 10% FBS for 48 h prior to RT-PCR analysis. Total RNA was extracted
from the transfected U2OS cells using mirVana miRNA isolation kit
(Ambion, USA) according to the manufacturer’s protocol. The
quantification of mature miR-200c using TaqManMicroRNA assay included
a two-step RT-PCR. First, 10 ng of RNA from each sample was converted
to cDNA using TaqMan microRNA reverse transcription cDNA synthesis
kit (Applied Biosystems, California) with specific primers for miR-200c
or internal reference Z30 (Applied Biosystems, California) according
to the manufacturer’s protocol. Second, the PCR amplification
of target cDNA was performed on Rotor-Gene Q qPCR instrument (QIAGEN)
with specific primers for miR-200c or Z30 using AmpliTaq Gold enzymes.
The expression of miR-200c level was normalized to internal miRNA
reference Z30 using comparative CT method.
3
Thermal Stability Analysis of SAMHD1
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Thermal melt measurements were made using the GloMelt Thermal Shift protein stability kit from Biotium on a Qiagen Rotor-gene Q qPCR instrument. Solutions of 50 mM HEPES—pH 7.5, 50 mM KCl, 5 mM MgCl2, 0.5 mM TCEP, 3 μM SAMHD1, 1X GloMelt dye and 0/2 mM dGTPαS were subjected to a temperature gradient from 25 to 85°C at 0.5°C intervals. Each step in the gradient was held for three seconds and the fluorescence of the GloMelt dye was measured Rotor-gene Q green channel. Automatic gain optimization was performed all of the samples before execution of the temperature gradient with a signal limit of seven. Three replicates were recorded for each measured condition. Melt temperatures for each replicate were calculated using the program TSA-CRAFT (Supplemental Table S2) (29 (link)). To display differences in melt temperature between different SAMHD1 constructs, averaged traces were differentiated and normalized in Prism using 6th order polynomial smoothing considering five neighbors on each side.
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