The RNX-Plus solution (Phenol + Guanidine Isothiocyanate) for total RNA extraction was the product of SinaClon (Cat.no: PS4131). In brief, 24 h after treatment, the medium culture was removed from groups and the Total RNA of hASCs was extracted using the RNX- Plus Solution kit, according to the company manual. After purification and quantification, RNA was determined by measuring the optical density at 260 and 280 nm using Nanodrop (Nanodrop- ND-1000). PrimeScript reagent kit (Cat.no: RR037Q) for cDNA synthesis, was purchased from Takara Inc. Real Q Plus 2x and the first strand of cDNA was generated from 500 ng of extracted total RNA using the Takara PrimeScript reagent kit according to the protocol provided by the manufacturer. Master Mix Green High ROX was purchased from Ampliqon (Cat.no: A 235402). SYBER green and ROX were used as the reporter and reference dies respectively and the relative amount of mRNA for each target was normalized to the gene expression. Gene-specific primer sets used in this study are shown in
Primescript reagent kit
Manufactured by Takara Bio
Sourced in Japan, China, United States
The PrimeScript reagent kit is a collection of reagents designed for reverse transcription and polymerase chain reaction (RT-PCR) experiments. The kit includes components necessary for the synthesis of complementary DNA (cDNA) from RNA templates and subsequent amplification of specific gene sequences.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (57 mentions)
Trizol, by Thermo Fisher Scientific (22 mentions)
Sybr premix ex taq, by Takara Bio (15 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (14 mentions)
Sybr green premix ex taq, by Takara Bio (14 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (14 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (13 mentions)
Rneasy mini kit, by Qiagen (8 mentions)
Gdna eraser, by Takara Bio (7 mentions)
Rnaiso plus kit, by Takara Bio (7 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (6 mentions)
Sybr premix ex taq 2, by Takara Bio (6 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Rnaiso plus, by Takara Bio (5 mentions)
Cfx96 real time system, by Bio-Rad (5 mentions)
7900 fast real time pcr system, by Thermo Fisher Scientific (5 mentions)
Sybr green, by Takara Bio (5 mentions)
Purelink rna micro scale kit, by Thermo Fisher Scientific (5 mentions)
Sybr green master mix, by Thermo Fisher Scientific (5 mentions)
Sybr premix ex taq 2 kit, by Takara Bio (4 mentions)
187 protocols using primescript reagent kit
1
RNA Extraction and Quantification Protocol
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The RNX-Plus solution (Phenol + Guanidine Isothiocyanate) for total RNA extraction was the product of SinaClon (Cat.no: PS4131). In brief, 24 h after treatment, the medium culture was removed from groups and the Total RNA of hASCs was extracted using the RNX- Plus Solution kit, according to the company manual. After purification and quantification, RNA was determined by measuring the optical density at 260 and 280 nm using Nanodrop (Nanodrop- ND-1000). PrimeScript reagent kit (Cat.no: RR037Q) for cDNA synthesis, was purchased from Takara Inc. Real Q Plus 2x and the first strand of cDNA was generated from 500 ng of extracted total RNA using the Takara PrimeScript reagent kit according to the protocol provided by the manufacturer. Master Mix Green High ROX was purchased from Ampliqon (Cat.no: A 235402). SYBER green and ROX were used as the reporter and reference dies respectively and the relative amount of mRNA for each target was normalized to the gene expression. Gene-specific primer sets used in this study are shown inTable 1 . For statistical analysis, the results were presented as means ± SEM. Statistical differences between different groups were tested by One-way analysis of variance (ANOVA) using Graph Pad Prism software. P < 0.05 was determined as significant.
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The RNX-Plus solution (Phenol + Guanidine Isothiocyanate) for total RNA extraction was the product of SinaClon (Cat.no: PS4131). In brief, 24 h after treatment, the medium culture was removed from groups and the Total RNA of hASCs was extracted using the RNX- Plus Solution kit, according to the company manual. After purification and quantification, RNA was determined by measuring the optical density at 260 and 280 nm using Nanodrop (Nanodrop- ND-1000). PrimeScript reagent kit (Cat.no: RR037Q) for cDNA synthesis, was purchased from Takara Inc. Real Q Plus 2x and the first strand of cDNA was generated from 500 ng of extracted total RNA using the Takara PrimeScript reagent kit according to the protocol provided by the manufacturer. Master Mix Green High ROX was purchased from Ampliqon (Cat.no: A 235402). SYBER green and ROX were used as the reporter and reference dies respectively and the relative amount of mRNA for each target was normalized to the gene expression. Gene-specific primer sets used in this study are shown in
2
Quantifying miR-sc3 and Astn1 mRNA
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To determine miR-sc3 expression, a total amount of 50 ng RNA was reverse transcribed using a TaqMan ® MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) or Prime-Script reagent Kit (Takara, Dalian, China) according to manufacturer's instructions. To detect the mRNA level of Astn1, RNA samples were reverse transcribed to cDNA by using a Prime-Script reagent Kit (Takara) according to manufacturer's instructions. Quantitative real-time RT-PCR was performed using SYBR Green Premix Ex Taq (Takara) on an Applied Biosystems StepOne real-time PCR System. All reactions were run in triplicate. The relative expression was calculated using the comparative 2 -DDCt method.
3
Investigating XIAP-miR-431-5p Interactions
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Wild-type (WT) and mutant sequences of the 3' untranslated region (UTR) of XIAP with the putative miR-431-5p binding sites were synthesised and cloned into the pmirGLO vector (Promega). HFLS-RA cells were co-transfected with miR-431-5p mimics/mimics NC and WT/mutant constructs for 48 h and subjected to the dual-luciferase reporter assay (Promega) according to the manufacturers' instructions.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was isolated from synovial tissues and HFLS-RA cells using RNAiso Plus (Takara, Japan). We reverse transcribed 1 μg of RNA using the Mir-X TM miRNA First-Strand Synthesis kit and PrimeScript TM reagent kits (Takara) for the miRNA and mRNA samples, respectively. Gene expression was measured using the SYBR ® qRT-PCR kit (Takara) and normalised to that of GAPDH (mRNA) or U6 (miRNA). Relative expression of genes was calculated using the 2 -△△Ct method. The primers using for PCR were as follows: 5'-TGTCTTGCAGGCCGTCATGC -3' (forward) for hsa-miR-431-5p; 5'-ACACACTTCGGGTTTCACGA-3' (forward) and 5'-AAGTCCCTTCGTCTCCCTCA-3' (reverse) for XIAP; 5'-ATGTTGCAACCGGGAAGGAA-3' (forward) and 5'-AGGAAAAGCATCACCCGGAG-3' (reverse) for GAPDH.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was isolated from synovial tissues and HFLS-RA cells using RNAiso Plus (Takara, Japan). We reverse transcribed 1 μg of RNA using the Mir-X TM miRNA First-Strand Synthesis kit and PrimeScript TM reagent kits (Takara) for the miRNA and mRNA samples, respectively. Gene expression was measured using the SYBR ® qRT-PCR kit (Takara) and normalised to that of GAPDH (mRNA) or U6 (miRNA). Relative expression of genes was calculated using the 2 -△△Ct method. The primers using for PCR were as follows: 5'-TGTCTTGCAGGCCGTCATGC -3' (forward) for hsa-miR-431-5p; 5'-ACACACTTCGGGTTTCACGA-3' (forward) and 5'-AAGTCCCTTCGTCTCCCTCA-3' (reverse) for XIAP; 5'-ATGTTGCAACCGGGAAGGAA-3' (forward) and 5'-AGGAAAAGCATCACCCGGAG-3' (reverse) for GAPDH.
4
Investigating XIAP-miR-431-5p Interactions
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Wild-type (WT) and mutant sequences of the 3' untranslated region (UTR) of XIAP with the putative miR-431-5p binding sites were synthesised and cloned into the pmirGLO vector (Promega). HFLS-RA cells were co-transfected with miR-431-5p mimics/mimics NC and WT/mutant constructs for 48 h and subjected to the dual-luciferase reporter assay (Promega) according to the manufacturers' instructions.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was isolated from synovial tissues and HFLS-RA cells using RNAiso Plus (Takara, Japan). We reverse transcribed 1 μg of RNA using the Mir-X TM miRNA First-Strand Synthesis kit and PrimeScript TM reagent kits (Takara) for the miRNA and mRNA samples, respectively. Gene expression was measured using the SYBR ® qRT-PCR kit (Takara) and normalised to that of GAPDH (mRNA) or U6 (miRNA). Relative expression of genes was calculated using the 2 -△△Ct method. The primers using for PCR were as follows: 5'-TGTCTTGCAGGCCGTCATGC -3' (forward) for hsa-miR-431-5p; 5'-ACACACTTCGGGTTTCACGA-3' (forward) and 5'-AAGTCCCTTCGTCTCCCTCA-3' (reverse) for XIAP; 5'-ATGTTGCAACCGGGAAGGAA-3' (forward) and 5'-AGGAAAAGCATCACCCGGAG-3' (reverse) for GAPDH.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was isolated from synovial tissues and HFLS-RA cells using RNAiso Plus (Takara, Japan). We reverse transcribed 1 μg of RNA using the Mir-X TM miRNA First-Strand Synthesis kit and PrimeScript TM reagent kits (Takara) for the miRNA and mRNA samples, respectively. Gene expression was measured using the SYBR ® qRT-PCR kit (Takara) and normalised to that of GAPDH (mRNA) or U6 (miRNA). Relative expression of genes was calculated using the 2 -△△Ct method. The primers using for PCR were as follows: 5'-TGTCTTGCAGGCCGTCATGC -3' (forward) for hsa-miR-431-5p; 5'-ACACACTTCGGGTTTCACGA-3' (forward) and 5'-AAGTCCCTTCGTCTCCCTCA-3' (reverse) for XIAP; 5'-ATGTTGCAACCGGGAAGGAA-3' (forward) and 5'-AGGAAAAGCATCACCCGGAG-3' (reverse) for GAPDH.
5
Quantifying miRNA and mRNA Expression
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Total RNA was isolated from synovial tissues and HFLS-RA cells using RNAiso Plus (Takara, Japan). We reverse transcribed 1 μg of RNA using the Mir-X™ miRNA First-Strand Synthesis kit and PrimeScript™ reagent kits (Takara) for the miRNA and mRNA samples, respectively. Gene expression was measured using the SYBR® qRT-PCR kit (Takara) and normalized to that of GAPDH (mRNA) or U6 (miRNA). Relative expression of genes was calculated using the 2−△△Ct method. The primers using for PCR were as follows: 5′-TGTCTTGCAGGCCGTCATGC − 3′ (forward) for hsa-miR-431-5p; 5′-ACACACTTCGGGTTTCACGA-3′ (forward) and 5′-AAGTCCCTTCGTCTCCCTCA-3′ (reverse) for XIAP; 5′-ATGTTGCAACCGGGAAGGAA-3′ (forward) and 5′-AGGAAAAGCATCACCCGGAG-3′ (reverse) for GAPDH.
6
Isolation and Analysis of Retinal Microglia RNA
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Total RNA of the isolated CD11b/c+ retinal microglia from diabetes rats or BV2 were extracted using Trizol Reagent (Invitrogen, USA) according to the protocol. Reverse transcription assay was performed using PrimeScript™ reagent kits (TaKaRa, Japan) to obtain the cDNA. QRT-PCR, reactions were performed in LightCycler®480 PCR system (Roche, USA) using SYBR® Premix Taq™ kits (TaKaRa, Japan). The relative expression levels were analyzed using the 2-ΔΔCt method. Primers for each gene are shown in Supplemental Table 1
7
Quantitative Analysis of Inflammatory Markers in Rat Brain
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Rat brain tissue or microglia were treated with Trizol; the tissues were separated thoroughly with a homogenizer, and the mRNA was extracted using chloroform and isoamyl alcohol. The RNA sediment was dissolved in DEPC water (Takara, Shanghai, China), quantified using a spectrophotometer, and reverse transcribed into cDNA by using Prime Script reagent kits (Takara, Shanghai, China). PCR was performed using SYBR Green PCR kits (Takara, Shanghai, China) by a Step One Real-Time PCR System. Relative RNA Expression was normalized to β-actin and calculated.18 (link) The primers used in the study are listed as follows: β-actin (5ʹ-TTCGCCATGGATGACGATATC-3ʹ and 5ʹ- TAGGAGTCCTTCTGACCCATAC-3ʹ), Cox-2 (5ʹ-TTCCAGTATCAGAACCGCATTGCC-3ʹ and 5ʹ-CCGTGTTCAAGGAGGATGGAGTTG-3ʹ), IL-β (5ʹ-ATCTCACAGCAGCATCTCGACAAG-3ʹ and 5ʹ-CACACTAGCAGGTCGTCATCATCC-3ʹ), P2X7R (5ʹ-CCACTCTGCTGCCTTGTCGTTAC-3ʹ and 5ʹ-CTGGTATGCGGTTAGATGCGATGG-3ʹ), and TNF-α (5ʹ-GCATGATCCGAGATGTGGAACTGG-3ʹ).
8
qRT-PCR for Gene Expression Analysis
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qRT‒PCR was performed as previously described. Briefly, total cellular RNA was isolated using TRIzol (Invitrogen, 15,596,018) and reverse transcribed into cDNA using PrimeScript™ reagent kits (TaKaRa, RR036A). qRT‒PCR was performed by using TB Green Premix Ex Taq (Takara, RR820A) according to the manufacturer’s instructions on a 7500 real-time PCR detection system (Applied Biosystems, Carlsbad, CA). Data were normalized to GAPDH data from control samples, and relative expression levels were analyzed using the 2−∆∆ Ct method. The primers specific for each gene are listed in Table S2 .
9
Quantitative Gene Expression Analysis
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Total cellular RNA was isolated using TRIzol (Invitrogen, 15596018) and reverse transcribed into cDNA using PrimeScript™ reagent kits (TaKaRa, RR036A). Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed by using TB Green Premix Ex Taq (Takara, RR820A) according to the manufacturer’s instructions on a 7500 Real-Time PCR detection system (Applied Biosystems, Carlsbad, CA). Data were normalized to GAPDH data in control samples, and relative expression levels were analyzed using the 2−△△ Ct method. The primers for each gene are listed in Additional file 5 : Table S1.
10
Quantification of Liver IL-1β mRNA Expression
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The liver mRNA was extracted using the BIOZOL kit reagent (Bioer, China) according to the instructions. The quality and quantity of total RNA were assessed by reading absorbance at 260/280 nm using a spectrophotometer (Nanodrop2000, Thermo, USA). cDNA measurement was accomplished using a PrimeScriptTM reagent kit (Takara Bio Inc. Japan) in accordance with the manufacturer’s instructions. Then, cDNA was amplified according to RT-qPCR using SYBR® Green PCR Master Mix in the presence of IL-1β primer (forward sequence 5′-CAACAAAAATGCCTCGTGCTG-3′ and reverse sequence 5′-TCGTTGCTTGTCTCTCCTTGTA-3′) and β-actin primer (forward sequence 5′-CGCAAATTACCCACTCCCGAC-3′ and reverse sequence 5′-GTAACCTCCCGTTCAGACCAC-3′). The primers were designed by Oligo 7.0 software and they were confirmed by Blast Nucleotide (NCBI). PCR carried out in primary denaturation at 95 °C for 10 min. RT-q PCR was performed in 40 cycles (including secondary denaturation at 95 °C for 15 sec, annealing at 60 °C for 20 sec, and extension at 72 °C for 25 sec). β-Actin gene was used as an internal control gene to control the expression of mRNA. Further, the ΔΔCT method was used for the analysis of gene expression.
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