Total RNA from collected cells or tissues were extracted using TRIzol reagent. The growth media were removed from the culture dish. Approximately 1 ml of TRIzol was directly added to each well, and the cell lysis was achieved by repeatedly grinding. The cells were incubated at room temperature for 5 min. Afterward, 250 μl of chloroform was added to each sample. The samples were incubated for 10 min at room temperature and subsequently centrifuged at 13,500 g at 4°C for 15 min. The samples were divided into three layers. The aqueous phase was mixed with 500 μl of isopropyl alcohol at room temperature for 35 min. The samples were centrifuged at 13,500 g at 4°C for 10 min, and removed the supernatant. The RNA pellet was gently washed with 1 ml of ethanol, centrifuged at 10,600 g for 5 min at 4°C, and resuspended in 10 μl of RNA-free water. NanoDrop 8000 (Thermo, U.S.A.) was used to determine the concentration and purity of the extracted RNAs. The relative expression levels of mRNAs and miRNAs were quantified by the mirVana™ quantitative reverse transcription-PCR (qRT-PCR) miRNA Detection Kit and quantitative RT-PCR with SYBR Green I (Applied Biosystems, Foster City, CA). The relative expression levels were calculated based on Ct values and were normalized to the U6 or GAPDH levels of each sample, respectively.
Qrt pcr mirna detection kit
Manufactured by Thermo Fisher Scientific
Sourced in Canada, United States
The QRT-PCR miRNA Detection Kit is a laboratory equipment designed for the detection and quantification of microRNA (miRNA) expression levels using real-time reverse transcription-polymerase chain reaction (qRT-PCR) technology. The kit provides the necessary reagents and protocols to facilitate the analysis of miRNA expression in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Sybr green 1, by Thermo Fisher Scientific (1 mentions)
Nanodrop 8000, by Thermo Fisher Scientific (1 mentions)
Mirvana qrt pcr primer set, by Thermo Fisher Scientific (1 mentions)
Iq5 real time pcr system, by Bio-Rad (1 mentions)
Sybr green mix kit, by Thermo Fisher Scientific (1 mentions)
Cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Trizol plus rna purification kit, by Thermo Fisher Scientific (1 mentions)
Reverse transcription kit, by Takara Bio (1 mentions)
Abi 7500 real time pcr instrument, by Thermo Fisher Scientific (1 mentions)
Rnase free dnase, by Qiagen (1 mentions)
7 protocols using qrt pcr mirna detection kit
1
Total RNA Extraction and Quantification Protocol
2
Quantitative Analysis of miRNA Expression
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Total RNA was isolated from cultured cells using TRIzol reagent (Invitrogen). For miRNA expression analysis, qRT-PCR was performed using qRT-PCR miRNA Detection Kit and mirVana qRT-PCR Primer Sets (Applied Biosystems) according to the manufacturer's protocols. Human U6 served as an internal control. For quantitative PCR, cDNA was mixed with 2 × SYBR Green PCR Master Mix (Applied Biosystems) and various sets of gene-specific primers and then subjected to RT-PCR quantification using the iQ5 Real-Time PCR system (Bio-Rad). All reactions were performed in triplicate. The relative amounts of mRNA were calculated by using the comparative Ct method. The results are presented as fold-change of each miRNA.
3
Quantifying miR-21-3p and ECM Genes
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Total RNA was isolated from tissues and cells using Trizol reagent (Invitrogen, Carlsbad, USA) according to the manufacturer's protocols. Total RNA (1 μg) was used for synthesizing rst-strand cDNA using cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, USA). qRT-PCR was performed with SYBR Green Mix kit (Applied Biosystems) according to the manufacturer's instructions. The relative RNA levels were calculated using the ∆∆Ct method. GAPDH levels served as an internal control. miR-21-3p expression was assessed using the qRT-PCR miRNA Detection Kit (Applied Biosystems), with U6 levels as an internal control. The primer sequences used for the ampli cation are shown as follows.
Human miR-21-3p: sense 5′-CGCGCCAACACCAGTCGATG-3′; antisense 5′-GTGCAGGGTCCGAGGT-3′mouse α-SMA: sense, 5′-GACGCTGAAGTATCCGATAGAACACG-3′; antisense 5′-CACCATCTCCAGAGTCCAGCACAAT-3′; mouse TGF-β1: sense, 5′-AGCGGACTACTATGCTAAAGAGGTCACCC-3′; antisense, 5′-CCAAGGTAACGCCAGGAATTGTTGCTATA-3′; mouse Col1α1: sense, 5′-GGAGGGCGAGTGCTGTGCTTT-3′; antisense, 5′-GGGACCAGGAGGACCAGGAAGT 3′; mouse Col4α1: sense, 5′-TGGTCTTACTGGGAACTTTGCTGC-3′; antisense, 5′-ACCCTGTGGTCCAACGACTCCTCTC-3′; mouse GAPDH: sense, 5′-CGACTTCAACAGCAACTCCCACTCTTCC-3′; antisense, 5′-TGGGTGGTCCAGGGTTTCTTACTCCTT-3′; human GAPDH: sense, 5′-GCTGGCGCTGAGTACGTCGTGGAGT-3′; antisense, 5′-CACAGTCTTCTGGGTGGCAGTGATGG-3′.
Human miR-21-3p: sense 5′-CGCGCCAACACCAGTCGATG-3′; antisense 5′-GTGCAGGGTCCGAGGT-3′mouse α-SMA: sense, 5′-GACGCTGAAGTATCCGATAGAACACG-3′; antisense 5′-CACCATCTCCAGAGTCCAGCACAAT-3′; mouse TGF-β1: sense, 5′-AGCGGACTACTATGCTAAAGAGGTCACCC-3′; antisense, 5′-CCAAGGTAACGCCAGGAATTGTTGCTATA-3′; mouse Col1α1: sense, 5′-GGAGGGCGAGTGCTGTGCTTT-3′; antisense, 5′-GGGACCAGGAGGACCAGGAAGT 3′; mouse Col4α1: sense, 5′-TGGTCTTACTGGGAACTTTGCTGC-3′; antisense, 5′-ACCCTGTGGTCCAACGACTCCTCTC-3′; mouse GAPDH: sense, 5′-CGACTTCAACAGCAACTCCCACTCTTCC-3′; antisense, 5′-TGGGTGGTCCAGGGTTTCTTACTCCTT-3′; human GAPDH: sense, 5′-GCTGGCGCTGAGTACGTCGTGGAGT-3′; antisense, 5′-CACAGTCTTCTGGGTGGCAGTGATGG-3′.
4
VEGF Signaling Pathway Activation in Oral Cancer
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After treated with Populus euphratica extract, the changes of the VEGF signaling pathway activation levels in the oral cancer cells was determined with real time RT-PCR. This experiment was performed totally in accordance with the instructions with only a little change 13) . Shortly, the oral cancer cells in the logical growth phage were collected and seeded into the 6-well plates at the destiny of 5×10 5 cells/well in DMEM-FBS medium. After 24 h incubation in an incubator at the 37℃, 5 ％ CO 2 condition, the cells were collected and the total RNA in the cells were extracted with TRIZOL reagent. The quality of the total RNA was evaluated using the OD260/OD280 ratio, and the cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Cambridge, MA, USA) . The PCRs were conducted using the qRT-PCR miRNA Detection Kit (Invitrogen) : 95℃ for 15 min, followed by 40 cycles of denaturation at 95℃ for 5 s, annealing at 55℃ for 30 s and extension at 72℃ for 30 s. Each experiment was performed in triplicate and the relative quantification was analyzed by the 2 -ΔΔCt method. All the results were presented as mean±SD.
5
Quantifying Hippo Pathway mRNA in OPM-2 Cells
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The mRNA expressions of hippo pathway in OPM-2 cancer cells after treatment with compounds 1 and 2 were measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR) according to the protocol (22 (link)). Total RNA in OPM-2 cells was extracted using TRIzol™ Plus RNA Purification Kit (Invitrogen) according to the manufacturer’s instructions, and then the quality of RNA was evaluated using the OD260/OD280 ratio. The cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). The PCR primer sequences in this experiment are listed in Table 2 . The PCRs were conducted using the qRT-PCR miRNA Detection Kit (Invitrogen): 95°C for 15 min, followed by 40 cycles of denaturation at 95°C for 5 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s. Each experiment was performed in triplicate and the relative quantification was analyzed by the 2–ΔΔCt method.
Primers and si-RNA sequences.
| Name | Sequence |
|---|---|
| yap | |
| gapdh | |
| yap-siRNA | GGCAGACUGAAUUCUAAAUUUUUCCGUCUGACUUAAGAUUUA |
| Control-siRNA | UUCUCCGAACGUGUCACGUTTACGUGACACGUUCGGAGAATT |
6
Quantifying Gene Expression in Glioma
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Total RNA was isolated from glioma cells or tissues using TRIzol (Invitrogen, Carlsbad, CA) based on the guiding of manufacturer's specification. RNA quantity was determined by a NanoDrop2000 spectrophotometer (Thermo Scientific, Waltham, MA). For qRT‐PCR, RNA (1 μg) was reverse transcribed to cDNA using a reverse transcription kit (Takara, Dalian, China). SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA) and miRNA qRT‐PCR detection kit were used for the PCR on an ABI7500 Real‐time PCR instrument (Applied Biosystems). GAPDH was measured as an internal control. The primers for LINC00511, miR‐124‐3p and CCND2 were listed in Table S1 . Relative expression was calculated using the 2‐ΔΔCT method.
7
RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted using Trizol (Invitrogen) and treated with RNase-free DNase (Qiagen). Power SYBR qPCR and miRNA qRT-PCR detection kit (Applied Biosystems) were performed in a qRT-PCR detection system (ABI, USA) according to the manufacturer’s protocols.
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