Total RNA was isolated from the spinal cord using an RNeasy Micro Kit (Qiagen). For complementary DNA (cDNA) synthesis, the reverse transcriptase reaction was performed using a Prime Script first-strand cDNA Synthesis Kit (Takara Bio). Quantitative RT-PCR was performed using primers specific to the genes of interest (Additional file 4 : Table S1) and SYBR Premix Dimmer Eraser (Takara Bio). The data were normalized to glyceraldehyde-3-phosphate dehydrogenase. RT-PCR was conducted using a Thermocycler (Biometra, Göttingen, Germany) and products were detected by electrophoresis and ethidium bromide staining.
Sybr premix dimmer eraser
Manufactured by Takara Bio
Sourced in Japan
SYBR Premix Dimmer-Eraser is a real-time PCR reagent. It is designed for the detection and quantification of DNA sequences.
Automatically generated - may contain errors
4 protocols using sybr premix dimmer eraser
1
Quantitative RT-PCR for Spinal Cord Gene Expression
2
Gene Expression Analysis of Monocyte/Macrophage Cells
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Total RNA was isolated from the FACS-sorted monocyte/macrophage obtained from the spinal cord tissue and peripheral blood at 12 hpi using the RNeasy Micro Kit (74,004, Qiagen, Hilden, Germany) and THP-1 cells at 4 h after LPS treatment using the RNeasy Mini Kit (74,104, Qiagen). For complementary DNA (cDNA) synthesis, a reverse transcriptase reaction was performed using a PrimeScript first-strand cDNA Synthesis Kit (6110A, Takara Bio, Otsu, Japan). Quantitative real-time PCR was performed using primers specific to the genes of interest (Supplementary Table 1) and a SYBR Premix Dimmer-Eraser (RR091A, Takara Bio, Shiga, Japan). Data were normalized to the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
3
Quantitative RT-PCR Gene Expression Analysis
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Total RNA was isolated from LF cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and cDNA was synthesized from the total RNA using a PrimeScript reverse transcriptase (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions. Quantitative RT-PCR was performed using 20 μl reaction mixture with primers specific to the genes of interest (Table 1 ) and SYBR Premix Dimmer-Eraser (TaKaRa) [20 (link)]. The mRNA levels in each sample were normalized to those of glyceraldehyde-3-phosphate dehydrogenase mRNA (n = 5 per group).
4
Transcriptional Analysis of Hypertrophied Ligamentum Flavum
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Total RNA was isolated from LF cells using an RNeasy Mini Kit (Qiagen, Hilden, Germany), and cDNA was synthesized from the total RNA using PrimeScript Reverse Transcriptase (Takara, Tokyo, Japan) according to the manufacturer's instructions. RT-qPCR was performed using 20 mL of reaction mixture with primers specific to the genes of interest (Table 2) and SYBR Premix Dimmer-Eraser (Takara). 18 The mRNA levels in the human samples (n Z 15 per group) and the mouse samples (n Z 8 per group) were normalized to those of glyceraldehyde-3-phosphate dehydrogenase mRNA. In the analysis of human samples, the severely hypertrophied LF specimens were divided into dorsal and dural layers as described previously. 19 RT-qPCR was performed after obtaining samples (1 mm in width) from each layer of the severely hypertrophied LF and from the dural layer of the nonhypertrophied LF.
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