Total RNA was isolated from BMSCs using the RNAios Plus reagent (TaKaRa, Japan) and was reverse transcribed to cDNA using the PrimeScript TM RT Reagent Kit with gDNA Eraser (TaKaRa). Target genes were amplified with cDNA, specific primers and NovoStart®SYBR qPCR SuperMix plus (Novoprotein, China) in a CFX96 Touch Real-Time PCR detection system (Bio-Rad Laboratories, USA). PCR amplification was performed in triplicate at 95 °C for 15 s, followed by 40 cycles at 95 °C for 5 s, at 60 °C for 30 s, at 95 °C for 15 s, at 60 °C for 15 s, and at 95 °C for 15 s. The relative expression levels of the target gene were normalized to GAPDH levels and determined by the 2−ΔΔCt method. The information on the primers is shown in Table S1 (Supplemental file 1).
Rnaios plus reagent
Manufactured by Takara Bio
Sourced in Japan
RNAios Plus reagent is a solution designed for the extraction and purification of total RNA from various biological samples. It utilizes a guanidinium-based method to effectively isolate RNA, including small RNAs, from cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt reagent kit with gdna eraser, by Takara Bio (4 mentions)
Light cycle real time pcr machine, by Roche (2 mentions)
Nano 300 ultramicro spectrophotometer, by Allsheng (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Lightcycler 480 2, by Roche (2 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
Novostart sybr qpcr supermix plus, by Novoprotein (1 mentions)
Sybr green qpcr kit, by Accurate Biology (1 mentions)
Rnase free dnase 1, by Thermo Fisher Scientific (1 mentions)
Reverse transcription kit, by Promega (1 mentions)
Lightcycler nano system, by Roche (1 mentions)
Sybr green, by Yeasen (1 mentions)
Tianscript 2 rt kit, by Tiangen Biotech (1 mentions)
Tb green, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
M mlv reverse transcriptase, by Promega (1 mentions)
Genechip human transcriptome array 2, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
9 protocols using rnaios plus reagent
1
Quantifying Gene Expression in BMSCs
2
RNA Extraction and qRT-PCR Analysis of PDLSCs
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PDLSCs were washed with cold phosphate buffer solution (PBS), and then total RNA was extracted by RNAios Plus reagent (Takara, Japan) following the manufacturer's instructions. Then, the total RNA was reverse transcribed to cDNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara). qRT-PCR was carried out using a SYBR Green q-PCR kit (Accurate Biology, China) according to the manufacturer's instructions. Finally, the 2-ΔΔ (ct) method was used to compare the values of different groups. The primer sequences are shown in Table 1 .
3
Quantitative RT-PCR Gene Expression Analysis
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Total RNA was extracted using RNAios PLUS reagent (Takara), and 1 μg RNA was reverse‐transcribed by oligo (dT) primers using a reverse transcription kit (Promega) after digestion with RNase‐free DNaseI (Fermentas). The qRT‐PCR assay was performed in triplicate with SYBR Green I Master reagent and the Light Cycler Nano system (Roche). Actin was used as the internal control for normalization. The primers used in this study are listed in Table S1 .
4
Gene Expression Analysis in RAW 264.7 Cells
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Total RNA was extracted from RAW 264.7 cells using RNAios Plus reagent (Takara, Kusatsu, Japan) and RNA concentration was measured with a Nano‐300 ultramicro‐spectrophotometer (All Sheng, Zhejiang, China). TIANScript II RT Kit (KR107‐02, Tiangen, Shanghai, China) was used to synthesize cDNA. qPCR was performed using SYBR Green (11198ES08, YEASEN, Shanghai, China) on a Light Cycle® Real‐Time PCR machine (Roche, Switzerland). The primers used were synthesized by Sangon Biotech (Shanghai, China) (Table S2 ). GAPDH housekeeping gene was used as internal control. Transcript levels of target genes were calculated using the 2−∆∆Ct method.
5
RNA Extraction, cDNA Synthesis, and qRT-PCR
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The total RNA was isolated from cells by RNAios Plus reagent (Takara) according to the instruction of manufacture. After the purity and concentration of RNA were measured by NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA), RNA was reverse transcribed to cDNA by PrimeScript ™ RT reagent Kit (Takara). The qRT-PCR was performed with TB green (Takara) by Roche LightCycler®480II. GAPDH was used as the internal control. The sequences of primers are listed in supplemental file 1.
6
Quantifying RNA Expression in Melanoma Cells
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Total RNA was extracted from B16–F10 melanoma cells treated with 8-MOP for 12 h using RNAios Plus reagent (Takara, Kusatsu, Japan). RNA concentration was measured with a Nano300 ultramicro-spectrophotometer (All Sheng, China). M-MLV reverse transcriptase (Promega, China) was used to synthesize cDNA following the manufacturer's protocol. qPCR was performed by using SYBR Green on a Light Cycle® Real-Time PCR machine (Roche, USA). GAPDH gene was used as an internal control for normalization. Transcript levels of target genes were calculated relative to a reference gene using the 2–ΔΔCt method [24 (link)]. Primers used in this study were synthesized by Sangon Biotech (China) and are shown in Supplementary Table S2 .
7
Osteogenic Differentiation of DPSCs
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Three DPSCs clones isolated from three different individuals were prepared for microarray analyses. In the experimental group, all DPSCs clones were cultured in osteogenic induction medium for 7 days, then the total RNA of cells was isolated by RNAios Plus reagent (Takara, Tokyo, Japan). In the control group, the total RNA was collected after DPSCs clones were cultured in cell culture medium for 7 days. The GeneChip Human Transcriptome Array 2.0 (Affymetrix, Santa Clara, CA, USA) and GCBI platform (GMINIX Informatics Ltd. Co, Shanghai, China) were used to analyze all RNA samples, then the differentially expressed mRNAs and lncRNAs between the experimental group and the control group were screened according to requirements of fold change (FC)≥1.5 and p < 0.05.
8
Quantitative RT-PCR Gene Expression Analysis
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Total RNA was isolated from cells by RNAios Plus reagent (Takara) according to the instructions. Total 1 μg RNA (in 10 μl reaction volume) was reverse transcribed to cDNA using PrimeScript ™ RT reagent Kit with gDNA Eraser (Takara). QRT-PCR was carried out in a reaction volume of 10 μl of SYBR® Premix Ex Taq™ (Takara) by Roche LightCycler® 480II as follows: an initial denaturation at 95℃ for 30 s, followed by 55 cycles of 95℃ for 5 s, 60℃ for 35 s, and extension at 72℃ for 1 min, finally at 40℃ for 30 s. The results were normalized against the internal control GAPDH and calculated by the 2-△△Ct method (△△Ct=(CT target-CT GAPDH) cocuture-(CT target-CT GAPDH) control). The expression levels of COL I, Runx2 and OCN were analyzed and primers used in this study were as follows:
COL I forward 5'-GCTGATGATGCCAATGTGGTT-3',
COL I reverse 5'-CCAGTCAGAGTGGCACATCTTG-3',
Runx2 forward 5'-GTTTCACCTTGACCATAACCGT-3',
Runx2 reverse 5'-GGGACACCTACTCTCATACTGG-3',
OCN forward 5'- AATCCGGACTGTGACGAGTTG-3',
OCN reverse 5'- CAGCAGAGCGACACCCTAGAC-3'.
COL I forward 5'-GCTGATGATGCCAATGTGGTT-3',
COL I reverse 5'-CCAGTCAGAGTGGCACATCTTG-3',
Runx2 forward 5'-GTTTCACCTTGACCATAACCGT-3',
Runx2 reverse 5'-GGGACACCTACTCTCATACTGG-3',
OCN forward 5'- AATCCGGACTGTGACGAGTTG-3',
OCN reverse 5'- CAGCAGAGCGACACCCTAGAC-3'.
9
Quantification of gene expression in BMSCs
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Total RNA was isolated from 2 M BMSCs and 18 M BMSCs using the RNAios Plus reagent (Takara) and was reverse transcribed to cDNA using the PrimeScript TM RT Reagent Kit with gDNA Eraser (Takara). The cDNAs were amplified by PCR with the following thermocycler conditions: 95 ℃ for 2 min, then 40 cycles of 95 ℃ for 15 s, and 60 ℃ for 1 min. GAPDH was used as an internal control to quantify and normalize the results. The information on the primers is shown in Table S1 (Additional file 1 ).
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