SH-SY5Y cells treated as described above were harvested. The total RNA was extracted as recommended by the manufacturer (RNeasy® Lipid Tissue Handbook, Qiagen, Germantown, MD, USA). Complementary DNA (cDNA) was synthesized from 2 μg RNA using the GoScript™ Reverse Transcriptase (Promega, Madison, WI, USA). Primer sequences (Table 2 ) were obtained from Invitrogen Corporation. PCR reactions were prepared using Quantitect SYBR Green master mix (Qiagen, Germantown, MD, USA) and carried out using a Real-Time PCR Detection Systems (Bio-Rad, Hercules, CA, USA) CFX96™ Real-Time System. The real-time PCR was optimized to run with conditions of the initial incubation at 95 °C for 5 min, denaturation at 94 °C for 15 s, annealing at 59 °C for 30 s, and extension at 72 °C for 30 s with a single fluorescence measurement and up to 38 cycles. Expression levels of target mRNAs were normalized to beta actin (relative quantification) with the ΔΔCT correction.
Rneasy lipid tissue handbook
Manufactured by Qiagen
Sourced in United States
The RNeasy® Lipid Tissue Handbook is a laboratory equipment product from Qiagen. It is designed for the isolation and purification of total RNA from lipid-rich tissues.
Automatically generated - may contain errors
Lab products found in correlation
Applied biosystems 7500 real time pcr system, by Thermo Fisher Scientific (4 mentions)
Primescript rt reagent kit manual, by Takara Bio (2 mentions)
Qiazol lysis reagent, by Qiagen (2 mentions)
Rneasy lipid tissue mini kit, by Qiagen (2 mentions)
Sybr green system, by Roche (2 mentions)
Goscript reverse transcriptase, by Promega (1 mentions)
Real time pcr detection system, by Bio-Rad (1 mentions)
Quantitect sybr green master mix, by Qiagen (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
3 protocols using rneasy lipid tissue handbook
1
Quantitative Gene Expression Analysis in SH-SY5Y Cells
2
Prdm12 Mutant RNA Expression Analysis
3
Prdm12 Mutant RNA Expression Analysis
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DRGs were collected from mutant and wild-type animals at E12.5 from both Prdm12 lines (Prdm12+/+, Prdm12+/−, Prdm12−/−, Prdm12+/LZand Prdm12LZ/LZ). mRNA was isolated using the RNeasy® Lipid Tissue Mini Kit (QIAGEN, Hilden, Germany), following the manufacturer’s instructions. Tissue was homogenized in QIAzol® Lysis Reagent using a TissueLyser II and optional DNase digestion was performed according to the RNeasy® Lipid Tissue Handbook (QIAGEN). Reverse transcription (RT)-PCR was performed using PrimeScript RT reagent kit manual (Takara Bio Inc., Kusatsu, Japan) employing equal amounts of RNA among samples. qPCR was performed based on the SYBR® Green System (F. Hoffmann-La Roche AG, Basel, Switzerland) using the Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific, Inc.). Relative mRNA expression of TrkA/Ntrk1, Ngn1, Neurod1, Pou4f1, Rbfox3, and Prdm12 were analyzed. The obtained expression levels were normalized using the expression of Actb as reference gene (the Prdm12+/+ values for normalized mRNA expression of each gene were arbitrarily set to 1).
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