Total RNA was extracted from cells using the RNeasy Mini kit (Qiagen, Valencia, CA). The cDNA was transcribed from 500 ng of total RNA using iScript cDNA Synthesis Kits (Bio-Rad, Hercules, CA), according to the manufacturer's protocol. The cDNA was used as the template for real-time PCR detection using TaqMan Technology on an Applied Biosystems 7000 sequence detection system (Applied Biosystems, Foster City, CA). Expression of BIRC5, CTGF, Gli2 genes and endogenous control gene b-glucuronidase (GUSB) were detected using the primer and probe sequences commercially available (Applied Biosystems) and analyzed using Relative Quantification Software (Applied Biosystems).
Applied biosystems 7000 sequence detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Applied Biosystems 7000 Sequence Detection System is a real-time PCR instrument used for gene expression analysis, genotyping, and other nucleic acid quantification applications. It features a thermal cycler and a detector that measures fluorescence signals during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (11 mentions)
Primer and probe sequences, by Thermo Fisher Scientific (6 mentions)
Relative quantification software, by Thermo Fisher Scientific (5 mentions)
Power sybr green rt pcr reagent, by Thermo Fisher Scientific (5 mentions)
Iscript cdna synthesis kit, by Bio-Rad (5 mentions)
Premix ex taq kit, by Takara Bio (4 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Turbo dna free, by Thermo Fisher Scientific (2 mentions)
Taqman reverse transcription reagents kit, by Thermo Fisher Scientific (2 mentions)
Primescript rt polymerase, by Takara Bio (2 mentions)
Specific primers, by Sangon (2 mentions)
Taqman technology, by Thermo Fisher Scientific (2 mentions)
High pure rna isolation kit, by Roche (2 mentions)
Revert ace kit, by Toyobo (1 mentions)
Taqman microrna assay, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq 2, by Takara Bio (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
24 protocols using applied biosystems 7000 sequence detection system
1
Quantitative PCR Gene Expression Analysis
2
Quantifying NEAT1 and miR-107 Expression
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Total RNA was extracted by using Trizol reagent (Invitrogen, Carlsbad, CA, USA). 2 μg of total RNA were reversely transcribed to cDNA by using High Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA, USA). Quantitative PCR was performed using Power SYBR Green RT-PCR Reagents (Applied Biosystems, Foster City, CA, USA). Primers were as follows: NEAT1 forward, 5′-CTTCCTCCCTTTAACTTATCCATTCAC-3′; reverse, 5′-CTCTTCCTCCACCATTACCAACAATAC-3′; miR-107 forward, 5′-ATGATGAGCAGCATTGTACAGG-3′; reverse, 5′-GCAGGGTCCGAGGTATTC-3′. All reactions were carried out on the Applied Biosystems 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Relative mRNA and lncRNA levels were normalized against β-actin and U6 snRNA, respectively, and calculated using the 2-ΔΔCt method.
3
Quantifying HMGB1 and miR-142-3p Expressions
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Total RNA was extracted from purified mononuclear cells and cultured cells using TriZol Reagent (Takara, Dalian, China). First-strand cDNA was synthesized from these RNA samples using Revert Ace kit (TOYOBO, Osaka, Japan). For the analyses of HMGB1 and miR-142-3p expressions, RT-PCR was performed using a SYBR Premix Ex Taq II (Takara) and TaqMan microRNA assay (Applied Biosystems, Foster City, CA) on the Applied Biosystems 7000 Sequence Detection System (Applied Biosystems), respectively. The specific primers used were as follows: HMGB1 forward 5′-CTGTCCATTGGTGATGTTGC-3′, reverse 5′- CTGATAGCCTGCTCCAGGTC-3′; miR-142-3p forward 5′-TGCGGTGTAGTGTTTCCTACTT-3′, reverse 5′-CCAGTGCAGGGTCCGAGGT-3′; GAPDH forward 5′-TCGGAGTCAACGGATTTGG-3′, reverse 5′-CATGGGTGGAATC ATATTGGA-3′. The relative gene expressions of miR-142-3p and HMGB1 mRNA were calculated by the 2−ΔΔCt method, and GAPDH level was used as the internal control.
4
Quantification of EIF5A2 in ATC
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Total RNA was isolated from ATC cells and the frozen ATC tissues using RNeasy Protect Kit (Life Technologies, Shanghai, China) and transcribed into cDNA using Superscript II Reverse Transcriptase (Invitrogen) according to the manufacturer’s instruction. The primer pairs for RT-PCR were EIF5A2: 5′-CCCTGCTGACAGAAACTGGT-3′ and 5′-TTGCACACATGACAGACACC-3′; GAPDH: 5′-AATCCCATCACCATCTTCCAGGAG-3′ and 5′-GCATTGCTGATGATCTTGAGGCTG-3′. RT-PCR was performed using an Applied Biosystems 7000 sequence detection system (Applied Biosystems, Foster City, CA, USA). Data were analyzed by the ΔΔ cycle threshold method.
5
RNA Isolation and Quantitative Real-Time PCR
6
Quantitative Analysis of HULC, miR-200a-3p, and ZEB1
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Total RNA was extracted by RNAiso Plus (Takara, Dalian, China). The reaction mixture (20 μl) containing 1 μg of total RNA was reversely transcribed to cDNA by using PrimeScript RT-polymerase (Takara, Dalian, China). Quantitative PCR was performed on the cDNA using specific primers (Sangon, Shanghai, China) for HULC (F:5‘-TCATGATGGAATTGGAGCCTT-3′, R: 5′-CTCTTCCTGGCTTGCAGATTG-3′), miR-200a-3p (5′- AACACTGTCTGGTAACGATGTCGT-3′), ZEB1 (F: 5′- CAGCTTGATACCTGTGAATGGG-3′, R: 5′-TATCTGTGGTGTGGGACT-3′) and GAPDH (F: 5′-CAGCCAGGAGAAATCAAACAG-3′, R: 5′-GACTGAGTACCTGAACCGGC-3′). All reactions were performed using the Applied Biosystems 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Relative expression levels were calculated as ratios normalized against those of GAPDH. Comparative quantification was determined using the 2−Δ ΔCt method.
7
ABCG2 Expression in Sorted Cells
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Total RNA was extracted from SP and non-SP cells using the RNeasy Mini Kit (Qiagen, Valencia, CA). The cDNA was transcribed using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). The cDNA was used as a template for real-time PCR using the Applied Biosystems 7000 sequence detection system (Applied Biosystems, Foster City, CA). Expression of ABCG2 and endogenous control gene β-glucuronidase (GUSB) were detected using the commercially available primer and probe (Applied Biosystems) and analyzed using Relative Quantification Software (Applied Biosystems).
8
RNA Isolation and Quantitative Real-Time PCR
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Total RNA was isolated from islet or islet and BM co-cultures using Trizol reagent (Invitrogen, catalog no# 10296-028). Isolated RNA was treated with Turbo DNA-free (Ambion, Catalog no# AM1907) to remove genomic DNA cDNA was synthesized from 500ng of total RNA using Applied Biosystems TaqMan® reverse transcription reagents kit (Applied Biosystems, catalog no.: N808-0234) with random hexamers in a total volume of 100 μl according to the manufacturer's instructions. cDNA was stored at -20°C for downstream PCR. Quantitative real time PCR was performed using Superarray SYBR green PCR kit (Superarray, Catalog no.: PA-012) in a total volume of 25 μl on Applied Biosystems 7000 sequence detection system (Applied Biosystems). The signal levels were normalized with GAPD for quantitative real-time PCR. Conditions for the PCR reaction were 10 min at 95°C, and then 40 cycles each consisting of 15s at 95°C, 60s at 60°C.
9
Quantitative RT-PCR for Gene Expression
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TRIzol reagent (Invitrogen, USA) was employed to isolate the total RNA from 16HBE cells. The extracted RNA was then reverse transcribed into cDNA adopting a Reverse Transcription Kit (Takara, Dalian, China). SYBR Premix Ex Taq (Takara, Dalian, China) was used for RT-qPCR. The reaction was performed on an Applied Biosystems 7000 Sequence Detection System (Applied Biosystems). The detection results for mRNAs were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). U6 acted as the endogenous reference for miRNAs. Expression fold changes were calculated using the 2−ΔΔCt method [36 (link)].
10
RNA Extraction and qPCR Analysis
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TRIzol reagent (Invitrogen) was employed to extract total RNA from HRMECs. Next, the extracted RNA was reverse transcribed to cDNA with High Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA, United States). Quantitative PCR was conducted with Power SYBR Green RT-PCR Reagents (Applied Biosystems, Foster City, CA, United States). All reactions were performed on Applied Biosystems 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, United States). Data were processed using the 2–ΔΔCt method (Livak and Schmittgen, 2001 (link)) normalized to U6 or GAPDH. Primers were listed as follows:
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