Total RNA was isolated from handpicked islets, livers and hypothalamus using an RNase-free DNase and RNeasy Kit (Qiagen, Valencia, CA). The concentration of total RNA was measured by a NanoDrop One (Applied Biosystems). It was confirmed that the A260/A280 ratio was 1.8–2.1 and the A260/A230 ratio was more than 2.0 in extracted total RNA. cDNA was prepared using High Capacity cDNA Reverse Transcription Kits (Applied Biosystems) and was subjected to qPCRquantitative PCR using Sybr Green Gene Expression Assays (7900 Real-Time PCR System; Applied Biosystems) with THUNDERBIRD qPCR Master Mix (TOYOBO). The primers are listed in Table S2 . A DNA microarray was performed using an Agilent-074809 Sure Print G3 Mouse GE8 × 60K Microarray (GPL21163) (Agilent, Santa Clara, CA, USA). The quality of total RNA was measured by a Bioanalyzer (Agilent), and the samples showing an RNA integrity number (RIN) of 7.0 or higher were used for microarray analysis. Data were analyzed using Genespring GX software (Agilent). The microarray data can be found at GEO: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE147269 .
Sybr green gene expression assays 7900 real time pcr system
Manufactured by Thermo Fisher Scientific
The Sybr Green Gene Expression Assays are a set of reagents designed for use with the 7900 Real-Time PCR System. The assays enable the detection and quantification of gene expression levels in a sample using the SYBR Green fluorescent dye technology.
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Lab products found in correlation
Thunderbird qpcr master mix, by Toyobo (2 mentions)
Rneasy kit and rnase free dnase, by Qiagen (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop one, by Thermo Fisher Scientific (1 mentions)
Genespring gx software, by Agilent Technologies (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
2 protocols using sybr green gene expression assays 7900 real time pcr system
1
RNA Isolation and Gene Expression Analysis
2
Quantitative PCR Analysis of Pancreatic Islet RNA
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Total RNA was isolated from pancreatic islets using an RNase-Free DNase and RNeasy Kit (QIAGEN, Valencia, CA). cDNA was prepared using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems) and was diabetesjournals.org/diabetes Li and Associates subjected to quantitative PCR using SYBR Green Gene Expression Assays (7900 Real-Time PCR System; Applied Biosystems) with THUNDERBIRD qPCR Master Mix (TOYOBO). Each quantitative reaction was performed in duplicate. Data were normalized according to the b-actin or GAPDH level. The primers described in Supplementary Table 2 were used for amplification.
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