Total RNA was isolated by using the TRIzol reagent extraction kit (Invitrogen), according to the manufacturer`s instructions. RNA was subsequently reverse transcribed to cDNA by using the high capacity cDNA archive kit (ABI). The mRNA levels were measured by real time PCR using the ABI PRISMTM 7900HT Sequence Detection System (Applied Biosystem).
Trizol reagent extraction kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The TRIzol reagent extraction kit is a laboratory product designed for the isolation and purification of RNA, DNA, and proteins from a wide range of biological samples. It utilizes a monophasic solution of phenol, guanidine isothiocyanate, and other proprietary components to effectively lyse cells and solubilize nucleic acids and proteins.
Automatically generated - may contain errors
Lab products found in correlation
Abi 7500 real time pcr system, by Thermo Fisher Scientific (3 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (3 mentions)
Sybr green qpcr kit, by Thermo Fisher Scientific (2 mentions)
Sybr qpcr kit, by Takara Bio (1 mentions)
7300 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr kit, by Thermo Fisher Scientific (1 mentions)
Superscript first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Abi prism tm 7900ht sequence detection system, by Thermo Fisher Scientific (1 mentions)
High capacity cdna rt kit, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Qiagen (1 mentions)
Primer premier 5, by Premier Biosoft (1 mentions)
Lightcycler 480 system, by Roche (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (1 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
8 protocols using trizol reagent extraction kit
1
Total RNA Extraction and qRT-PCR Analysis
2
Gene Expression Analysis of DLX6-AS1 and miR-497-5p
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The cell or tissue sample was collected after treatment with different procedures. The total RNAs were extracted with a TRIzol reagent extraction kit (Invitrogen, Karlsruhe, Germany). Reverse transcription was performed with the SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific). Quantitative assay of gene expressions was performed using an SYBR qPCR Kit (Takara, Osaka, Japan) and ABI 7500 real-time PCR system (Thermo Fisher Scientific, CA, USA). The gene expressions were normalized to GAPDH and calculated using the ΔCT method. The specific primer sequences were as follows: DLX6-AS1 – F: 5′-AGTTTCTCTCTAGATT-GCCTT-3′ and R: 5′-ATTGACATGTTAGTGCCCTT-3′, miR-497-5p – F: 5′-CCTTCAGCAGCA CACTGTGG-3′ and R: 5′-CAGTGCAGGGT CCGAGGTAT-3′, and GAPDH – F: 5′-GGGAAATTCAACGGCACAGT-3′ and R: 5′-AGATGGTGATGGGCTTCCC-3′.
3
Quantitative RT-PCR Analysis of EMT Markers
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Total RNA was extracted from the cultured SW480 cells using a TRIzol Reagent Extraction kit (Invitrogen, Carlsbad, CA, USA) according to the protocol provided by the manufacturer. Primers for cdx2, wnt3a, snail, twist, e-cadherin, n-cadherin, vimentin, and gapdh are listed in Table 1 . A BeyoRT™ cDNA kit (Beyotime, Shanghai, China) was used to conduct the reverse transcription of cDNA. Amplifications were performed using the 7300 Real-Time PCR system (Applied Biosystems, Waltham, MA, USA) and SYBR Green PCR kit (Invitrogen, Carlsbad, CA, USA). The reaction conditions were: pre-denaturation for 4 min at 95°C followed by 30 cycles of denaturation for 30 s at 95°C, annealing for 30 s at 57°C, and extension for 30 s at 72°C. Fold-changes of transcription levels for the targeted genes were determined using the 2−ΔΔCt method, with gapdh as the internal reference.
4
Quantitative Analysis of Gene Expression
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The total RNA from the cells or tissue samples was extracted with a TRIzol reagent extraction kit (Invitrogen, Karlsruhe, Germany). The reverse transcription was performed with the SuperScript III First-Strand Synthesis system (Invitrogen, Karlsruhe, Germany). Quantitative assay of gene expressions was performed by an SYBR Green qPCR Kit (Finnzymes, Espoo, Finland) and an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA). The gene expressions were normalized to the β-actin and calculated using the 2–ΔΔCT method. The specific primer sequences were designed and synthesized by GenePharma. The primers for ATP2B1-AS1, miR-330-5p, TLR4, GAPDH, and U6 were listed as follows: ATP2B1-AS1 (Forward, 5′-GCTCTGACGTCTGTGTTTCCA-3′; Reverse, 5′ -AAGTGAAGGGCGTCCCACT-3′); miR-330-5p (Forward, 5′ -GTCTCTGGGCCTGTGTC-3′; Reverse, 5′-TCCTCCTCTTCC TCTCCATT-3′); TLR4 (Forward, 5′-GCAGTTTCTGAGCAG TCGT-3′; Reverse, 5′-CCTCCCACTCCAGGTAAGT-3′); GAPD H, (Forward, 5′-AGGTCGGTGTGAACGGATTTG-3′; Reverse, 5′-GGGGTCGTTGATGGCAACA-3′); U6, (Forward, 5′-ACCC TGAGAAATACCCTCACAT-3′; Reverse, 5′-GACGACTGAGC CCCTGATG-3′).
5
RNA Extraction, RT-qPCR Gene Expression
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The total RNA from tissue samples was extracted with a TRIzol reagent extraction kit (Invitrogen). The reverse transcription was performed with the SuperScript First-Strand Synthesis system (Invitrogen). Quantitative assay of gene expressions was performed by an SYBR Green qPCR Kit and an ABI 7500 real-time PCR system (Applied Biosystems). The gene expressions were normalized to the GAPDH and calculated using the 2-ΔΔCT method. The specific primer sequences were designed and synthesized by GenePharma (Shanghai, China).
6
Gene Expression Analysis by qPCR
7
Quantitative Analysis of SPOP Expression
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Total RNA was extracted from tissue lysate using a TRIzol reagent extraction kit (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) followed by RT using the High Capacity cDNA Reverse Transcription kit (Qiagen, Inc., Valencia, CA, USA) in accordance with the manufacturer's protocol. The Roche Light Cycler 480 system (Roche Diagnostics, Burgess Hill, UK) was used for qPCR analysis. Primer pairs designed by Primer Premier 5.0 (Premier Biosoft International, Palo Alto, CA, USA) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China), the sequences of which were as follows: SPOP forward, 5′-TGACCACCAGGTAGACAGCG-3′ and reverse, 5′-CCCGTTTCCCCCAAGTTA-3′. The GAPDH gene served as an internal control. The sequences of the primers for GAPDH were as follows: GAPDH forward, 5′-AACTTCCGTTGCTGCCAT-3′ and reverse, 5′-TTTCTTCCACAGGGCTTTG-3′. The PCR system (25 µl) comprised 1 µl of cDNA, 12.5 µl of 2X Fast EvaGreen™ qPCR Master mix (Biotium Inc., Freemont, CA, USA), 1 µl of primers (10 µM) and 10.5 µl of RNase/DNase-free water. The following thermocycling conditions were maintained: 96°C for 2 min; 40 cycles at 96°C for 15 sec; and 60°C for 1 min. Each sample was performed in triplicate, and the average value was computed. For comparative expression of SPOP, the 2−∆∆Cq method (17 (link)) was used as an indication of relative changes.
8
Gene Expression Analysis by qRT-PCR
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Total RNA was extracted using a TRIzol™ Reagent Extraction kit (catalog# 15596018, Invitrogen), and cDNA was synthesized by reverse transcription using a High-Capacity cDNA Reverse Transcription Kit (catalog# 4368814, Applied Biosystems, Foster City, CA, USA). The primer sequences are listed in Table 1. qRT-PCR was performed on a 7500 Fast Real-Time PCR System (catalog# 4351106, Applied Biosystems, Foster City, CA, USA) using PowerUp™ SYBR™ Green Master Mix (catalog# A25742, Applied Biosystems, Foster City, CA, USA). U6 and GAPDH (glyceraldehyde-phosphate dehydrogenase) were used as independent internal references. The differential gene mRNA expression between the experimental and control groups was calculated using the 2 DDCT method.
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