Total RNA was extracted to determine the expression of CDR1as, miR-7, EGFR, PIK3CD, CCNE1, and RAF1 by using TRIzol® reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. A260/A280 nm >1.8 was qualified for quantitative analysis. Reverse transcription reaction was carried out to synthesize cDNA. qPCR was performed on 7500 Real-Time PCR detection system (Thermo Fisher Scientific). The expression levels of genes were estimated via 2−ΔΔCt method and were normalized to GAPDH and U6 for gene expression. The primer of genes used in this study are listed in Table 1 .
7500 real time pcr detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan, Germany
The 7500 Real-Time PCR Detection System is a laboratory instrument designed for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. It is capable of performing precise and sensitive nucleic acid detection and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (43 mentions)
Primescript rt reagent kit, by Takara Bio (19 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (13 mentions)
Trizol, by Thermo Fisher Scientific (11 mentions)
Rneasy mini kit, by Qiagen (10 mentions)
Sybr green master mix, by Roche (9 mentions)
Superscript first strand synthesis kit, by Thermo Fisher Scientific (7 mentions)
Reverse transcription system, by Takara Bio (7 mentions)
Quantitect sybr green pcr kit, by Toyobo (6 mentions)
Sybr premix ex taq, by Takara Bio (6 mentions)
Power sybr green pcr master mix, by Roche (5 mentions)
Nuclease free water, by Thermo Fisher Scientific (4 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Faststart universal sybr green master mix, by Roche (3 mentions)
Tri reagent, by Molecular Research Center (2 mentions)
Revertra ace qpcr rt kit, by Toyobo (2 mentions)
Reverse transcriptase kit, by Promega (2 mentions)
Primescript rt reagent kit perfect real time, by Takara Bio (2 mentions)
Sybr premix ex taq kit, by Takara Bio (2 mentions)
Trizol, by Takara Bio (2 mentions)
87 protocols using 7500 real time pcr detection system
1
Quantitative Analysis of Gene Expression
2
RNA Extraction and qPCR Analysis in Mice
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Total RNA was extracted from the indicated tissues of mice by using TRI Reagent (Molecular Research Center) or Sepasol I Super G (Nacalai Tesque), and cDNAs were synthesized with the RevertraAce qPCR RT Kit (Toyobo). To remove residual DSS, we prepared mRNAs from the colon of mice treated with DSS and then purified mRNAs by LiCl precipitation as described previously.79 (link) qPCR analysis was performed with the 7500 Real-Time PCR detection system with SYBR green method of the target genes and murine Hprt as an internal control with 7500 SDS software (Thermo Fisher Scientific). The primers used in this study are shown in Table S1 .
3
Differential Gene Expression in rBMSCs
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Total RNA was harvested from rBMSCs cultured in different BD2 concentrations
for 3 days using the EZ-press RNA purification kit (EZBioscience,
Shanghai, China), and complementary DNA (cDNA) reverse transcription
was performed using the PrimeScript RT reagent kit (Takara, Dalian,
China). The qPCR analysis was performed using Power SYBR Green PCR
Master Mix (Applied Biosystems, Foster City, CA) and a 7500 Real-Time
PCR detection system (Thermo Fisher Scientific, Waltham). The primer
sequences used, including for collagen I (Col1), osteocalcin (Ocn),
osteopontin (Opn), runt-related transcription factor 2 (Runx2), and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were reported earlier.46 (link) The relative mRNA levels measured were normalized
to the GAPDH expression level.
for 3 days using the EZ-press RNA purification kit (EZBioscience,
Shanghai, China), and complementary DNA (cDNA) reverse transcription
was performed using the PrimeScript RT reagent kit (Takara, Dalian,
China). The qPCR analysis was performed using Power SYBR Green PCR
Master Mix (Applied Biosystems, Foster City, CA) and a 7500 Real-Time
PCR detection system (Thermo Fisher Scientific, Waltham). The primer
sequences used, including for collagen I (Col1), osteocalcin (Ocn),
osteopontin (Opn), runt-related transcription factor 2 (Runx2), and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were reported earlier.46 (link) The relative mRNA levels measured were normalized
to the GAPDH expression level.
4
Quantifying VEGF-A Expression in hADSCs
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Total RNA from cultured hADSCs were extracted by TRIzol reagent (Invitrogen, California, USA) at 24 h following transfection, and the reverse transcription was employed using PrimeScript RT reagent kit (Takara Bio Inc., Otsu, Japan) as previously reported [14 ]. Real-time PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster, CA, USA) according to the protocol. The following primers were used: VEGF-A, forward primer, 5′-TTGCAGGTTGGTT CCCAGAGG-3′ and reverse primer, 5′-TCGGCTTGTCACATCTGAGGG-3′; β-actin, forward primer, 5′-GGGACCTGACTGACTACCTC-3′; and reverse primer, 5′-TCAT ACTCCTGCTTGCTGAT-3′. qPCR detection was carried out on a 7500 Real-Time PCR Detection System (Thermo Scientific, Waltham, MA, USA), and relative expression was determined using the 2-∆∆CT method.
5
Gene Expression Analysis by qPCR
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Total RNA was extracted with Quick-RNA™ MiniPrep kit (Zymo Research, Irvine, CA, USA). cDNA was reversely transcribed in a total volume of 20 μl reaction mixture containing RNA, first-strand buffer, DTT, dNTP, gene-specific reverse primers, SuperScript IV Reverse Transcriptase (Invitrogen), and RNaseOUT™ recombinant ribonuclease inhibitor (Invitrogen) in RNase-free H2O. cDNA was amplified by a 7500 Real-time PCR Detection System (Thermo Fisher Scientific) using a Fast SYBR Green Master Mix (Applied Biosystems, Waltham, MA, USA) containing forward and reverse primers. Target mRNA expression was normalized against Actb mRNA. The primers (forward and reverse) used for qPCR are as follows: Faslg: 5′-GCAGAAGGAACTGGCAGAAC-3′ and 5′-TTAAATGGGCCACACTCCTC-3′; Bcl2: 5′-GTCCTTCAGGTGTGGGATTT-3′ and GAGCAAATGCAGCCACAATAC-3′; Bax: 5′-AGGATGCGTCCACCAAGAAGCT-3′ and 5′-TCCGTGTCCACGTCAGCAATCA-3′; Actb: 5′-GGGAATGGGTCAGAAGGACT-3′ and 5′-CTTCTCCATGTCGTCCCAGT-3′.
6
Quantifying mRNA Levels in Liver and Immune Cells
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Total RNA was extracted from liver tissues, sorted hepatocytes, and various immune cells using the RNeasy® Mini Kit (74106; Qiagen, Venlo, Netherlands) according to the manufacturer's instructions and then reverse-transcribed into cDNA using a PrimeScript™ RT Master Mix (RR036A; Takara, Kyoto, Japan) or SMARTScribe™ Reverse Transcriptase (639538; Takara). qRT-PCR was performed using TB Green® Premix Ex Taq™ II (RR820A; Takara) and respective primers (listed in Table S5 ) on a 7500 Real-Time PCR Detection System (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The levels of mRNAs were determined using the 2‒ΔΔCt method and expressed as the ratio of the level of expression of the target gene to that of the housekeeping gene β-actin.
7
Purification and qPCR Analysis of RNA
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Total RNAs were extracted from the indicated tissues of mice by using TRI Reagent (Molecular Research Center) or Sepasol II Super (Nacalai Tesque), and cDNAs were synthesized with the RevertraAce qPCR RT Kit (Toyobo). To remove residual DSS, mRNAs prepared from the colon of mice treated with DSS were further purified by LiCl precipitation as described previously42 (link). Quantitative polymerase chain reaction (qPCR) analysis was performed with the 7500 Real-Time PCR detection system with CYBR green method of the target genes and murine Hprt an internal control with 7500 SDS software 2.3 (Thermo Fisher Scientific). The primers used in this study are shown in Supplementary Table 3 .
8
qRT-PCR Analysis of Fibrosis Markers in LX-2 Cells
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Trizol reagent (Thermo Fisher Scientific) was used to isolate total RNA from LX-2 cells. A reverse transcriptase polymerase chain reaction kit (RT-PCR) was used to reverse-transcribe RNA (2 µg) to obtain cDNA. A NanoDrop 8,000 spectrophotometer was used to quantify the RNA. PCR cycling procedures were as follows: 25°C for 5 minutes, 37°C for 60 minutes, and 70°C for 5 minutes. A 7500 real-time PCR detection system from Thermo Fisher Scientific was used in this experiment. Primers were purchased from Sangon Biotech (Shanghai, China). Collagen I: forward, 5′-TGGCCAAGAAGACATCCCTGAAGT-3′; reverse, 5′-ACATCAGGTTTCCACGTCTCACCA-3′. Fibronectin: forward, 5′-CCATCGCAAACCGCTGCCAT-3′; reverse, 5′-AACACTTCTCAGCTATGGGCTT-3′. α-smooth muscle actin (α-SMA): forward, 5′-ACTG AGCGTGGCTATTCCTCCGTT-3′; reverse, 5′-GCAGTGGCCATCTCATTTTCA-3′. GAPDH: forward, 5′-AAGAAGGTGGTGAAGCAGGC-3′; reverse, 5′-TCCACCACCCTGTTGCTGTA-3′. Target mRNA was normalized to GAPDH. GAPDH served as an endogenous control. Relative expression changes were determined using the 2−ΔΔCt method.
9
RNA Extraction and qPCR Analysis
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Total RNA was extracted using TRIzol reagent (Accurate Biotechnology, Hunan, China) and converted into cDNA (Evo M-MLV RT Premix for qPCR; Accurate Biotechnology). The miRNA RNAs were converted into cDNA using a miRNA 1st strand cDNA synthesis kit (Accurate Biotechnology, Hunan, China). β-Actin and U6 were used as reference controls for mRNAs, lncRNAs, and miRNAs. Primers were designed by Accurate Biotechnology (Hunan, China) (Table S2 ). qPCR was performed using the SYBR® Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology, Hunan, China) and the Applied Biosystems 7500 Real-Time PCR Detection System. The data were analyzed using the 2−ΔΔCt (Livak) relative expression method. All experiments were repeated three times.
10
Quantitative Analysis of Embryonic and Postnatal Gene Expression
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Total RNA was isolated from pups of E18.5 and P0 using the TRIzol reagent (Invitrogen) as previously described.63 (link) Reverse transcription was carried out utilizing the Evo M-MLVRT Premix (Applied Biosystems, Foster City, CA, USA). Quantitative RT–PCR was conducted with SYBR Green Pro Taq HS (Accurate Biotechnology (human) Co., Ltd) and the 7500 Real-Time PCR Detection System (Applied Biosystems, Foster City, CA, USA). The primer sequences are as follows: Gapdh, 5’-CCACTCTTCCACCTTCG-3’ and 5’-GTGGTCCAGGGTTTCTTAC-3’; Col1a1, 5′-TAGGCCATTGTGTATGCAGC-3′ and 5′-ACATGTTCAGCTTTGTGGACC-3′; Osx, 5′-ATGGCGTCCTCTCTGCTTG-3′ and 5′-TGAAAGGTCAG CGTATGGCTT-3′; Runx2, 5′-TCCACSSGGACAGAGTCAGATTACAG-3′ and 5′-CAGAAGTCAGAGGTGGCAGTGTCATC-3′; Alp, 5′-CGGGACTGGTACTCGGATAA-3′ and 5′-ATTCCACGTCGGTTCTGTTC-3′; Pck2, 5′-CCCTGACTGGACATGGGGAT-3′, and 5′-GGCAAAGCACTTCTTGCCCA-3′.
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