Total RNA was extracted using Trizol reagent (Invitrogen). 2 μg of total RNA from each sample was used for reverse transcription, the PCR was carried out as previously described. For real-time RT-PCR, SYBR Green real-time Master Mix (Toyobo, Japan) and the ABI7300 Real Time PCR were used (Applied Biosystems, USA). Target gene expression levels were normalized by calculating the Targets/18S expression ratio (2-ΔΔCt). Primers for real-time PCR were listed in the S1 Table .
Sybr green realtime master mix
Manufactured by Toyobo
Sourced in Japan, United States, China
SYBR Green Realtime Master Mix is a ready-to-use solution for real-time PCR applications. It contains SYBR Green I dye, DNA polymerase, dNTPs, and other necessary components for efficient and reliable real-time PCR amplification.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (21 mentions)
M mlv reverse transcriptase, by Promega (4 mentions)
Sybr primescript mirna rt pcr kit, by Takara Bio (3 mentions)
Cfx96 real time pcr system, by Bio-Rad (3 mentions)
Abi 7500 system, by Thermo Fisher Scientific (3 mentions)
Revertra ace rt pcr kit, by Toyobo (3 mentions)
Reverse transcription kit, by Takara Bio (2 mentions)
Iq5 multicolor detection system, by Bio-Rad (2 mentions)
Trizol rna extraction, by Thermo Fisher Scientific (2 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Applied biosystems 7500 sequence detection system, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Abi7300 real time pcr, by Thermo Fisher Scientific (1 mentions)
All in one rt mastermix, by Applied Biological Materials (1 mentions)
Lightcycler 480 system, by Roche (1 mentions)
Primescript rt reagent kit with oligodt primer, by Takara Bio (1 mentions)
Applied biosystems 7900 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Smartspec plus spectrophotometer, by Bio-Rad (1 mentions)
Revertra ace qpcr rt master mix, by Toyobo (1 mentions)
34 protocols using sybr green realtime master mix
1
Real-Time RT-PCR Gene Expression Analysis
2
Gene Expression Quantification by qRT-PCR
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Total RNA was extracted by TRIzol reagent (Invitrogen Inc., Carlsbad, CA, USA). Subsequently, the RNA was reversely transcribed to the cDNA. The M-MLV reverse transcriptase (Promega, Madison, WI, USA) was used for reverse transcription reaction. SYBR-Green Real-Time Master Mix (Toyobo, Tokyo, Japan) was applied for qRT-PCR reaction. Finally, the data were calculated with 2−ΔΔCt method.
HULC P1: 5′-AACCTCCAGAACTGTGAT-3′ and HULC P2: 5′-CATAATTCAGGGAGAAAG-3′
β-Actin P1: 5′-CTTCCTTCCTGGGCATGGAG-3′ and β-actin P2: 5′-GGAACGCTTCACGAATTTGC-3′
HULC P1: 5′-AACCTCCAGAACTGTGAT-3′ and HULC P2: 5′-CATAATTCAGGGAGAAAG-3′
β-Actin P1: 5′-CTTCCTTCCTGGGCATGGAG-3′ and β-actin P2: 5′-GGAACGCTTCACGAATTTGC-3′
3
Quantifying miR-186 and APAF1 Expression
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Total RNA was extracted from cSCC and control tissues, and miR-NC-, miR-186 mimic- or miR-186 inhibitor-transfected A-431 cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). mRNA was reverse transcribed into cDNA using the Revert Aid First Strand cDNA Synthesis kit (cat. no. K1622; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol, and the thermocycling conditions were 25°C for 5 min, 42°C for 60 min and 70°C for 10 min. qPCR was performed using SYBR-Green Real-Time Master mix (Toyobo Life Science, Osaka, Japan) following the manufacturer's protocol. The thermocycling conditions of qPCR were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. GAPDH and U6 were used as internal controls for APAF1 and miR-186, respectively. The primers used for the detection of miR-186 and APAF1 were as follows: miR-186, sense 5′-GCGGCGCAAAGAATTCTCCT-3′ and antisense 5′-GTGCAGGGTCCGAGGT-3′; APAF1, sense 5′-ATGGACACCTTCTTGGACGACAG-3′ and antisense 5′-TGTGGGGGCGGACAACTAA-3′; GAPDH, sense 5′-TGTTCGTCATGGGTGTGAAC-3′ and antisense 5′-ATGGCATGGACTGTGGTCAT-3′; U6, sense 5′-CGCTTCGGCAGCACATATACTA-3′ and antisense 5′-CGCTTCACGAATTTGCGTGTCA-3′. Primers were designed by Sangon Biotech Co., Ltd. (Shanghai, China). The relative expression of miR-186 and APAF1 were calculated and normalized using the 2−ΔΔCq method (25 (link)).
4
Quantitative Analysis of LINC00662 Expression
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Total RNA was isolated from tissue samples or cultured cells using TRIzol® reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. For this, 2 µg total RNA in 20 µL reaction mixture was reverse transcribed to complementary DNA (cDNA) using a Reverse Transcription Kit (TaKaRa, Dalian, China). Real-time quantitative PCR analyses were performed with SYBR green real-time Master Mix (TOYOBO, Japan) as described by the manufacturer. The PCR primers used for LINC00662: 5′-ACTAACAAGCTGGGTGCAGA-3′ (forward) and 5′-CCTCCTGGTCTGCGAGAAAT-3′ (reverse); for GAPDH: 5′-TGTTCGTCATGGGTGTGAAC-3′(forward) and 5′-ATGGCATGGACTGTGGTCAT-3′ (reverse). Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and data collection were performed on Applied Biosystems 7,500 Sequence Detection system (Thermo Fisher Scientific, Waltham, MA, USA). The thermocycling protocol for all experiments was 40 cycles of denaturation at 95°C for 20 seconds, annealing at 58°C for 20 seconds, and extension at 72°C for 20 seconds. The relative expression of LINC00662 was calculated and normalized using the 2−ΔΔCt method relative to GAPDH (internal control).
5
Quantitative PCR (qPCR) Analysis of Gene Expression
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Quantitative PCR (qPCR) was performed as previously described.50 (link) Total RNA was extracted using TRIzol reagent (Life Technologies, catalog number 15596-018). Equal amounts of total RNA from each sample were subjected to oligo (dT)-primed cDNA synthesis using 5× All-In-One RT MasterMix (Abm, catalog number G492). Reactions were run according to a standard protocol using SYBR Green Realtime MasterMix (TOYOBO, catalog number QPS-201) on a Roche Light Cycler 480 System (Roche Diagnostics). All results are presented in arbitrary units relative to human ACTIN mRNA expression. The specific primers used for qPCR are summarized in Supplementary Table S1 .
6
Quantitative Analysis of miR-139-5p and ROCK2 mRNA
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Total RNA was extracted from cultured cells or tissues using TRIzol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA quantity was measured by a SmartSpec Plus spectrophotometer (Bio-Rad, Hercules, CA, USA). RNA purity was evaluated by the A260/A280 ratio. The relative expression of miR-139-5p was detected using a SYBR PrimeScript miRNA RT PCR Kit (Takara, Dalian, P.R. China) in accordance with the manufacturer’s instructions, and U6 small nuclear RNA (snRNA) was used as an internal control. The specific primers of miR-139-5p and U6 were brought from Applied Biosystems (Foster City, CA, USA). For quantitative ROCK2 mRNA level, 2 μg of total RNA was reverse transcribed into cDNA using PrimeScript RT Reagent Kit with oligodT primer (Takara). Quantitative real-time PCR analysis was performed with SYBR Green Real-Time Master Mix (Toyobo, Co., Ltd, Osaka, Japan). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. qRT-PCR and data collection were performed on an Applied Biosystems 7900 Sequence Detection System (Applied Biosystems). Primers of ROCK2 and GAPDH were used in this study as described previously19 (link). The relative expression of miR-139-5p and ROCK2 mRNA was normalized to that of U6 or GAPDH using the 2−ΔΔCt method.
7
RT-qPCR Procedure for mRNA Quantification
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Total intracellular RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Total cDNA was synthesized by reverse transcription using the Rever Tra Ace qPCR RT Master Mix (Toyobo, Japan) according to the manufacturer’s protocols. To quantify mRNA expression, quantitative real-time PCR (RT-qPCR) was performed with CFX96 Real-time PCR System (Bio-Rad, Hercules, CA, USA) and SYBR Green Real-time Master Mix (Toyobo, Japan). The qPCR primers are listed in Table 1 . The selected gene mRNA levels relative to GAPDH have been calculated to obtain the relative mRNA expression.
8
Quantifying miR-152 and ROCK1 Expression
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Total RNA was isolated from the tissue and cell lines using TRIzol reagent (Invitrogen) following the manufacturer’s instruction. Total RNA was reversed transcribed to cDNA using M-MLV Reverse Transcription system (Promega, Madison, WI, USA). Real-time PCR for miR-152 expression was performed using SYBR green I mix (Takara, Dalian, P.R. China). To quantify ROCK1 mRNA expression, reverse transcription reaction and cDNA synthesis were performed according to the manufacturer’s instructions (Promega). Real-time PCR analysis was performed using SYBR Green Real-Time Master Mix (Toyobo, Osaka, Japan). Relative quantification of miR-152 and ROCK1 mRNA was achieved by normalizing U6 and β-actin, respectively.
9
Quantitative Real-Time PCR Analysis
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Total RNAs from tissue samples or cultured cells were extracted using TRIzol reagent (Invitrogen Inc., U.S.A.) and quantitated using a NanoDrop2000 (Thermo Scientific, U.S.A.). Total of 2 μg RNA was used for reverse transcription reaction and cDNA synthesis using M-MLV Reverse Transcriptase (Promega, U.S.A.). SYBR Green Real-time Master Mix (TOYOBO, Japan) was used for quantitative real-time PCR (qRT-PCR) analyses. The conditions of thermal cycling were illustrated as follows: 94°C for 2 min followed by 40 cycles at 94°C for 20 s, and at 58°C for 20 s. All primers were synthesized from Sangon Biotech (Shanghai, China). GAPDH was taken as the internal control. The LINC00982 primers were 5′-AAGTCGTGCTGAGTGTCTGG-3′ (forward) and 5′-CACAACGTGCCACGAACAAT-3′ (reverse). The GAPDH primers were 5′-TGTTCGTCATGGGTGTGAA-3′ (forward) and 5′-ATGGCATGGACTGTGGTCAT-3′ (reverse). Applied Biosystems 7500 Sequence Detection system (ABI, U.S.A.) was used for qRT-PCR and data collection. 2−ΔΔCT method was used to analyze the relative fold changes.
10
BDNF Expression Quantification by qRT-PCR
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Total RNA was extracted using Trizol reagent (Invitrogen). cDNAs were synthesized using the M-MLV reverse transcription kit (Promega) following the manufacturer’s instructions. Real-time PCR was performed on an ABI 7500 system (Applied Biosystems) using SYBR Green real-time Master Mix (Toyobo, Japan). The expression of each target gene was normalized to the Gapdh gene and calculated using the 2−ΔΔCt method. RNA was from three wells of one group and each sample has been examined in triplicate. Primer sequences were as follows: BDNF 5′-TGCAGGGGCATAGACAAAAGG-3′ (forward), 5′-CTTATGAATCGCCAGCCAATTCTC-3′ (reverse); Gapdh 5′-CATGTTCCAGTATGACTCCACTC-3′ (forward), 5′-GGCCTCACCCCATTTGATGT-3′ (reverse).
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