NHEKs were seeded on a 6‐well plate and cultured in KGM for overnight. The medium was changed to KBM, and cells were cultured for 24 h. The cells were then treated with or without Alpha‐KG for 24 h. Total RNAs were isolated in accordance with the standard operation method. Real‐time RT‐PCR reactions were performed by using a Thermal Cycler Dice® Real Time System III and TaKaRa SYBR® PrimeScriptTM RT‐PCR Kit (Perfect Real Time) (Takara Bio). The amount of mRNA expressions of filaggrin (FLG), serine palmitoyltransferase (SPT), and involucrin (IVL) was normalized by that of GAPDH used as an internal standard.
Takara sybr primescript rt pcr kit
Manufactured by Takara Bio
Sourced in China
The TaKaRa SYBR® PrimeScriptTM RT‐PCR Kit is a ready-to-use reagent system for reverse transcription and real-time PCR amplification. It utilizes SYBR® Green I dye for detection of amplified DNA.
Automatically generated - may contain errors
Lab products found in correlation
Rnaiso, by Takara Bio (1 mentions)
Dnase 1, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Primer premier 5, by Premier Biosoft (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Iq5 multicolor real time pcr detection system, by Bio-Rad (1 mentions)
Primescript rt kit, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
4 protocols using takara sybr primescript rt pcr kit
1
Measuring Epidermal Differentiation Markers
2
Quantitative Gene Expression Analysis in Lamprey
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The total RNA samples were extracted from lamprey tissues including supraneural myeloid bodies, gills, lymphocytes, hearts, livers and kidneys by using RNAiso (TaKaRa), and then treated with DNaseI (TaKaRa) to remove contaminated DNA. The reverse transcription was conducted using PrimeScript™ RT reagent Kit (TaKaRa). The real-time quantitative PCR (qPCR) was carried out with the TaKaRa SYBR ® PrimeScriptTM RT-PCR Kit (TaKaRa). The glyceraldehyde-3-phosphate-dehydrogenase gene (GAPGH) of L. japonica (accession number: KU041137) was used as an internal control. The primer sequences are Lja-CD81 Forward 5′-ACTCACCCTCCAACTTCCTC-3′, Reverse: 5′-CTCCAGGAAGT TCTTGGCAT-3′; GAPDH Forward: 5′-ACCAACTGCCTGGCTC CT-3′, Reverse: 5′-TCTTCTGCGTTGCCGTGT-3′. The amplification was performed under the following condition: initial denaturation at 95°C for 5 s to activate the DNA polymerase, followed by 40 cycles of 5 s at 95°C, 30 s at 57°C and 30 s at 72°C, and a final extension step at 65°C for 15 s. The 2 -ΔΔCT method was used to calculate the relative changes in gene expression from qPCR experiments [34] .
3
Quantitative Real-Time PCR Analysis of Gene Expression
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Total RNA from each sample was isolated by RNAiso Plus (Takara, China), then dissolved in water-DEPC and kept at a final concentration of 1000 μg/mL using Biophotometer Plus (Expander, Germany). Total RNA was reverse-transcribed by a Takara PrimeScript® RT reagent kit with gDNA eraser according to the manufacturer's manual (Takara, China). Primer sequences for qRT-PCR were designed using Primer Premier 5.0 (Premier Biosoft International, Palo Alto, CA, USA). qRT-PCR was performed using a Takara SYBR® PrimeScript™ RT-PCR kit (Takara, China). SYBR Green PCR cycling was performed on an iQ™ 5 Multicolor real-time PCR detection system (Bio-RAD, USA) using 20 μL samples with the following temperature program: 95°C for 10 s, followed by 40 cycles of 95°C for 15 s, and then annealing at 52°C for 30 s. The relative quantization of gene expressions were calculated and normalized to Actin. Three replicates were used for qRT-PCR.
4
Quantitative Gene Expression Analysis in Rice
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RNA was extracted using Trizol reagent (purchased from Invitrogen) from rice leaves. High-quality RNA was reverse transcribed into cDNA with Takara’s PrimeScriptTM RT Kit with a gDNA eraser (Takara, Dalian, China) according to the manufacturer’s instruction. Takara SYBR® PrimeScript™ RT-PCR kit (Takara, Dalian, China) was used to determine the expression of the particular genes. Quantitative expression analysis was carried out by the software Bio-Rad CFX Manager V2.0 software installed in the qPCR instrument. Actin was used as an internal control. The quantitative expression data were analyzed according to 2 ΔΔCT method proposed by Livak et al. [38 (link)]. All primer sequences used for qPCR are listed in Table S1 .
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