Total RNA was isolated from cultured primary adipocytes or from mouse tissues using QIAzol Lysis Reagent Protocol (QIAGEN) following the manufacturer's instructions. cDNA was synthesized from 1 μg of total RNA using iScript cDNA Synthesis Kit (BioRad). Quantitative RT-PCR was performed using iQ SybrGreen supermix on BioRad CFX97and analyzed as previously described [42] (link), [43] (link). 36B4, Hprt, and Gapdh served as controls for normalization. Primer sequences used for qRT–PCR analyses were listed in Supplementary Table 1 .
Qiazol lysis reagent protocol
Manufactured by Qiagen
QIAzol Lysis Reagent is a monophasic solution of phenol, guanidine isothiocyanate, and other proprietary components designed for the isolation of total RNA from various biological samples. The reagent can be used for the isolation of high-quality RNA from a wide range of sample types, including tissues, cells, and bacteria.
Automatically generated - may contain errors
5 protocols using qiazol lysis reagent protocol
1
Quantitative RT-PCR Analysis of Gene Expression
2
Quantitative RT-PCR from Mouse Tissues
3
Quantitative RT-PCR from Mouse Tissues
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Total RNA was isolated from mouse tissues using QIAzol Lysis Reagent Protocol (QIAGEN) following the manufacturer’s instructions. cDNA was synthesized from 1 μg of total RNA using iScript cDNA Synthesis Kit (BioRad). Quantitative RT-PCR was performed using iQ SybrGreen Supermix on a BioRad CFX97 thermocycler and analyzed as previously described (Livak and Schmittgen, 2001 (link); Schmittgen and Livak, 2008 (link)). 36B4, 18S and βM2 served as controls for normalization. Primer sequences used for qRT–PCR analyses were listed in Table S1 .
4
RNA Extraction and qRT-PCR Analysis
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Total RNA was isolated from mouse tissue using QIAzol Lysis Reagent Protocol (QIAGEN) following the manufacturer's instructions. cDNA was synthesized from isolated RNA using iScript cDNA Synthesis Kit (BioRad). Quantitative RT-PCR was performed using iQ SybrGreen supermix on a BioRad CFX97 RT-PCR system and analyzed as previously described [29] (link), [30] (link). 36B4, Hprt, and Gapdh served as controls for normalization. Primer sequences used for qRT–PCR analyses were listed in Supplementary Table 1 .
5
Quantitative RNA Expression Analysis
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Total RNA was isolated from scAT using QIAzol Lysis Reagent Protocol (QIAGEN) following the manufacturer’s instructions. cDNA was synthesized from 2 μg of total RNA using iScript cDNA Synthesis Kit (BioRad). RPL19 served as control for internal reference gene. Primer sequences used for qRT-PCR analyses were listed in Table S2 . Analyses of qRT-PCR products were performed with the Prism 7500 SDS software (Thermo Fisher Scientific). Relative quantification of mRNA amount was obtained by the by 2−(ΔΔCt) method.
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