Cultured astrocytes were treated with recombinant human BDNF (100 ng/ml; Peprotech, Seoul, Korea) or GDNF (100 ng/ml; R&D Systems) for 8 hr, and then RNA was extracted from cells with TRIzol reagent (Life Technologies). RNA isolation was performed using the RNeasy Mini Kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s instructions. cDNA synthesis was performed at 37°C for 120 min with 100 ng of RNA using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Waltham, MA, USA). Quantitative RT-PCR was performed using the one-step SYBR PrimeScript RT-PCR kit (Perfect Real Time; Takara Bio Inc., Shiga, Japan) according to the manufacturer’s instructions, followed by detection using an Applied Biosystems 7,500 Real-Time PCR system (Applied Biosystems). GAPDH was used as an internal control. The 2−ΔΔCt method was used to calculate relative changes in gene expression, as determined by real-time PCR experiments.
One step sybr primescript rt pcr kit perfect real time
Manufactured by Takara Bio
Sourced in Japan
The One-step SYBR PrimeScript RT-PCR kit (Perfect Real Time) is a reagent kit designed for real-time reverse transcription PCR (RT-PCR) analysis. It enables the detection and quantification of RNA targets in a single-tube reaction.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Abi prism 7000 sequence detection system, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Taqman universal master mix 2, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Applied biosystems 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Abi 7500 qpcr instrument, by Thermo Fisher Scientific (1 mentions)
Molpure cell rna kit, by Yeasen (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Dna engine tetrad peltier thermal cycler, by Bio-Rad (1 mentions)
Superscript 2, by Thermo Fisher Scientific (1 mentions)
Abi prism 7500 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
7500 fast rt pcr system, by Thermo Fisher Scientific (1 mentions)
7 protocols using one step sybr primescript rt pcr kit perfect real time
1
BDNF and GDNF Modulate Astrocyte RNA
2
Real-Time PCR Normalization with Internal Controls
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Real-time PCR was performed using a One-Step SYBR PrimeScript RT–PCR Kit (Perfect Real-Time; Takara Bio), followed by detection using an ABI Prism 7000 Sequence Detection System (Applied Biosystems). Normalization was performed using two internal controls, ribosomal protein lateral stalk subunit P0 (Rplp0) and tubulin α 1A (Tub1a), and a model-based variance and stability calculation (Vandesompele et al, 2002 (link); Andersen et al, 2004 (link)). The nucleotide sequences of the primers were based on published cDNA sequences (Table S14).
Table S14.
3
Spinal Cord mRNA Expression Analysis
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Mice were anesthetized and perfused with PBS to remove blood. Then, the lumbar spinal cord was rapidly dissected out. The spinal cord at L4-L5 was divided into two constituent parts; ipsilateral and contralateral spinal cord. Then, these were frozen in liquid nitrogen and homogenized in TRIzol reagent (Life Technologies, Carlsbad, CA, USA). Total RNA (2 mg) from each sample was reverse-transcribed into cDNA using a First Strand cDNA synthesis kit (MBI Fermentas, Hanover, Germany). Using the one-step SYBR® Prime-Script™ RT-PCR kit (Perfect Real-Time; Takara Bio Inc., Tokyo, Japan) and the ABI Prism® 7000 sequence detection system (Applied Biosystems, Foster City, CA, USA), real-time PCR was performed according to the manufacturer’s instructions. The 2−ΔΔCT method was used to calculate relative changes in gene expression,18 (link) and glyceraldehyde-3-phosphate dehydrogenase was used as a control. The primer nucleotide sequences are listed in Table S1 .
4
Quantifying FOSL1 and MMP13 mRNA Expression
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FOSL1 and MMP13 mRNA expression levels were determined using RT-qPCR. Briefly, total RNA was extracted from HT29 cells (1x106 cells) using the MolPure® Cell RNA kit (Shanghai Yeasen Biotechnology Co., Ltd.) according to the manufacturer's protocol. Complementary DNA was synthesized from the isolated RNA using the PrimeScript™ RT Reagent kit (Takara Bio, Inc.) according to the manufacturer's protocols. qPCR was performed using the One Step SYBR PrimeScript RT-PCR kit (Perfect Real Time; TakaRa Bio Inc.) and the ABI 7500 qPCR instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. The following thermocycling conditions were used: Initial denaturation at 95˚C for 10 min; 40 cycles of denaturation at 95˚C for 10 sec, annealing at 60˚C for 20 sec and elongation at 72˚C for 30 sec; and a final extension at 72˚C for 7 min. Relative mRNA expression levels were determined using the 2-ΔΔCq method (20 (link)). The experiments have been replicated for 3 times. Primers sequences were: FOSL1, forward 5'-TGACCACACCCTCCCTAACTC-3' and reverse 5'-CTGCTGCTACTCTTGCGATGA-3'; MMP13; forward, 5'-AACATCCAAAAACGCCAGAC-3' and reverse 5'-GGAAGTTCTGGCCAAAATGA-3', GAPDH, forward 5'-CCATGGGGAAGGTGAAGGTC-3' and reverse 5'-AGTGATGGCATGGACTGTGG-3'.
5
RNA Extraction and Real-Time PCR Analysis
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According to the manufacturer's instructions, total RNA was extracted from the other half of the resected SR muscle, using TRIzol reagent (Invitrogen). Total RNA (0.5 μg) was reverse-transcribed into cDNA, with Superscript II (Invitrogen) and oligo (dT) primers. Polymerase chain reaction (PCR) amplification was performed with a DNA Engine Tetrad Peltier Thermal Cycler (MJ Research, Waltham, MA, USA) at a temperature of 55–60°C for 20–30 cycles, with specific primer sets. For the analysis of the PCR products, 10 μl of each was electrophoresed on 1% agarose gel and detected under ultraviolet (UV) light. GAPDH was used as the internal control. Real-time PCR was done with the One Step SYBR® PrimeScript™ RT-PCR kit (Perfect Real-Time; Takara Bio Inc., Tokyo, Japan), and detection was done with the ABI Prism® 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA).
6
RNA Extraction and qPCR Analysis Protocol
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TRIzol reagent (Life Technologies Corporation, Carlsbad, CA, United States) was used to extract total RNA from ECs and GECs. RNA concentration and quality of each sample were determined with Nanodrop Spectrophotometer (ND-100) by the 260/280 nm ratio. The primers for MCM3AP-AS1, KLF5, and GAPDH were synthesized from Takara Bio (Japan). The primers for miR-211 and U6 were synthesized from the Applied Biosystems. The expression levels of MCM3AP-AS1, KLF5, and GAPDH were measured with One-Step SYBR PrimeScript RT-PCR Kit (Perfect Real Time; Takara Bio, Inc., Japan). MCM3AP-AS1: forward 5′-GCTGCTAATGGCAACACTGA-3′, reverse 5′-AGGTGCTGTCTGGTGGAGAT-3′; KLF5: forward 5′-GAACGTCTTCCTCCCTGACA-3′, reverse 5′-GGCAGTCGTTTCACTCTGGT-3′. MCM3AP: forward 5′-TGGGATTCAGACGCTTTCGC-3′, reverse 5′-TCCACAGCATCAATGGCACC-3′; TRPM1: forward 5′-GCAAACAGGTGGAGACTCAGC-3′, reverse 5′-ATTGGAATATCCGCCACCCTG-3′; GAPDH: forward 5′-CAGGAGGCATTGCTGATGAT-3′, reverse 5′-GAAGGCTGGGGCTCATTT-3′. The expression levels of miR-211 and U6 (Applied Biosystems, Foster City, CA, United States) were examined with High Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA, United States) and Taqman Universal Master Mix II (Life Technologies Corporation, Carlsbad, CA, United States). The relative quantification 2-ΔΔCt method was applied to calculate the gene expression.
7
Quantification of RNA Expression Levels
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Total RNAs were separated with Trizol reagent (Life Technologies, Carlsbad, CA, USA), following the manufacturer’s description. The RNA concentration and quality were determined using a Nanodrop Spectrophotometer (ND-100, Thermo Scientific, Waltham, MA). For measuring the levels of PIWIL1 (NM_004764.4), MEG3 (NR_033359.1), and RUNX3 (NM_001031680.2), One-Step SYBR PrimeScript RT-PCR Kit (Perfect Real Time) (Takara Bio) was used. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as endogenous control. The levels of piR-DQ593109 (hsa_piR_016792) and miR-330-5p (NR_029886.1) were detected by TaqMan MicroRNA Reverse Transcription kit and TaqMan Universal Master Mix II (Applied Biosystems). U6 was used as endogenous control. All qRT-PCR reactions were performed by the 7500 Fast RT-PCR System (Applied Biosystems, Foster City, CA, USA). Relative expression values were calculated using the relative quantification (2−ΔΔCt) method. Primers and probes used in this study are shown in Table S1 .
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