Cellular mRNA was extracted with Trizol (Invitrogen) and purified with the RNeasy Mini Kit (Qiagen). cDNA was prepared from mRNA using the Quantitect reverse transcription kit according to the manufacturer’s protocol (Qiagen). The efficiency of the primer pairs was evaluated using a standard curve and the stability of the expression of the RN18S1 or HPRT reference genes between treatments was evaluated. The primers for SCD-1 (137 bp) were forward- 5 ′-AGTTCTACACCTGGCTTTGG-3′ and reverse-5′-GTTGGCAATGATCAGAAAGAGC-3′, and those for SREBP-1c (164 bp) forward-5′-AGTCACTGTCTTGGTTGTTGA-3′ reverse-5′-GACCGACATCGAAGGTGAAG-3′. The primers for the reference genes are Forward 5′- GAGACTCTGGCATGCTAACTAG-3′ and reverse 5′-GGACATCTAAGGGCATCACAG-3′ and Forward 5′-TGCTGAGGATTTGGAAAGGG-3′ reverse 5′TTTATGTCCCCTGTTGACTGG-3′ for RN18S1 and HPRT, respectively. Gene expression was measured using 10 ng of cDNA by quantitative PCR (ABI 7500, Applied Biosystems) with Ssofast ™ Evagreen Supermix Low ROX (Bio-Rad).
Ssofast evagreen supermix low rox
Manufactured by Bio-Rad
SsoFast™ EvaGreen® Supermix Low ROX is a ready-to-use qPCR reaction mix that contains all the necessary components for real-time PCR amplification and detection using the EvaGreen dye. It is designed for high-throughput, fast, and sensitive quantification of DNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (1 mentions)
Abi 7500, by Thermo Fisher Scientific (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Biomark system, by Standard BioTools (1 mentions)
C1 single cell auto prep ifc for preamp, by Standard BioTools (1 mentions)
Fluidigm real time pcr analysis software, by Standard BioTools (1 mentions)
C1 single cell auto prep reagent kit, by Standard BioTools (1 mentions)
Ambion single cell to ct qrt pcr kit, by Thermo Fisher Scientific (1 mentions)
2 protocols using ssofast evagreen supermix low rox
1
Gene Expression Analysis of SCD-1 and SREBP-1c
2
Single-Cell RNA-seq Analysis Protocol
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Cells were isolated on C1 plates using the Biomark system (Fluidigm). Single cells were separated using a C1 Single‐Cell Auto Prep Reagent Kit (Fluidigm) and C1 Single‐Cell Auto Prep IFC for Preamp (10‐17 µm and 17‐25 µm) (Fluidigm). RNA was extracted and cDNA was synthesized and preamplified using an Ambion Single Cell‐to‐CT qRT‐PCR Kit (Thermo Fisher Scientific). These cDNA were preamplified using pooled primer sets for 47 genes. Primer sequences are listed in Table S1 . Products were amplified using individual primers, 48.48 Dynamic Array IFC EvaGreen (Fluidigm), GE 48.48 Dynamic Array DNA Binding Dye (Fluidigm) and SsoFast EvaGreen Supermix Low ROX (Bio‐Rad). The data were analyzed using the Fluidigm Real‐Time PCR Analysis software (Fluidigm). We also used program language R to analyze data in more detail using heatmap, violin plot and t‐distributed stochastic neighbor embedding (tSNE) analyses.
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