Total RNA was extracted from islets with miRNeasy Kits (Qiagen) and quantified using Nanodrop 1000 (Thermo Fisher Scientific). Relative mRNA expression was determined by RT-qPCR. cDNA was prepared using random primers and High Capacity Reverse Transcription Kit (#4368814) and expression was determined using TaqMan Universal PCR Master Mix I with TaqMan gene expression assays (Life Technologies). Amplification and Ct values were obtained using ViiA™ 7 Real-Time PCR System and software (Life Technologies). Relative gene expression was normalized against two reference genes. The following TaqMan primers were used: Atp2a2 Mm01201431_m1, Atp2a3 Mm00443898_m1, Sel1l Mm01326442_m1, Errfi1 (Mig 6) Mm00505292_m1; with reference genes Ppib Mm00478295_m1 and Hprt Mm00446968_m1. Data are given from 6 mice per condition and each experiment has 3 technical replicates (Fig 4 ).
Taqman universal pcr master mix 1
Manufactured by Thermo Fisher Scientific
TaqMan Universal PCR Master Mix I 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.
Automatically generated - may contain errors
Lab products found in correlation
Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
High capacity reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Mirneasy kit, by Qiagen (1 mentions)
Quantstudio 7 flex real time pcr system, by Thermo Fisher Scientific (1 mentions)
Mirneasy mini kit protocol, by Qiagen (1 mentions)
High capacity cdna reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Qiazol, by Qiagen (1 mentions)
2 protocols using taqman universal pcr master mix 1
1
Assessing Gene Expression in Mouse Islets
2
Quantitative PCR analysis of islet gene expression
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Pancreatic islets were lysed in Qiazol (Qiagen, Sollentuna, Sweden) and homogenized by vortexing. Total RNA was extracted using miRNeasy®Mini Kit protocol (Qiagen) and RNA concentration was measured on a NanoDrop (Thermo Scientific, Göteborg, Sweden). High Capacity cDNA Reverse Transcriptase Kit (Life Technologies, Stockholm, Sweden) was used to generate RT-PCR according to the manufacturer's protocol.
The QuantStudio™ 7 Flex Real-Time PCR System (Life Technologies) was used to performed qPCR according to the TaqMan®Universal PCR Master Mix I protocol (Life Technologies) using the following primers from TaqMan®Gene Expression Assays; SERCA2 (Mm01201431_m1), SERCA3 (Mm00443898_m1), Sel1l (Mm01326442_m1), CHOP (Mm01135937_m1), and Calb1 (Mm00486647_m1). Gene expression was normalized using Hprt (Mm004469_m1) and Ppia (Mm00478295_m1) as reference genes with the ΔΔCt method.
The QuantStudio™ 7 Flex Real-Time PCR System (Life Technologies) was used to performed qPCR according to the TaqMan®Universal PCR Master Mix I protocol (Life Technologies) using the following primers from TaqMan®Gene Expression Assays; SERCA2 (Mm01201431_m1), SERCA3 (Mm00443898_m1), Sel1l (Mm01326442_m1), CHOP (Mm01135937_m1), and Calb1 (Mm00486647_m1). Gene expression was normalized using Hprt (Mm004469_m1) and Ppia (Mm00478295_m1) as reference genes with the ΔΔCt method.
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