Total RNA was isolated from homogenized liver tissues using a TRIzol™ isolation kit (Takara Bio, Dalian, China) following the manufacturer's protocol. The cDNA was synthesized by using Primer Script RT kit (Takara Bio, Dalian, China). Prime Script™ RT reagent kits, along with SYBR Green Realtime PCR Master Mix and Permix Ex Taq (Takara Bio), according to the manufacturer's instructions. The primers for GAPDH, TNF-α, IL-10, TGF-β1, α-SMA, and Desmin were synthesized by Sangon Biotech (Shanghai, China). Real-Time PCR was operated on ABI Prism 7500 Sequence Detection System (BioRad, Life Science Research, Hercules, CA, USA). PCR conditions were as follows: one cycle at 95°C for 30 s, 40 cycles at 95°C for 5 s, at 64°C for 30 min. All reactions were performed in triplicate for each sample. The 2−ΔΔCT method was used to calculate relative concentration of each target by standardizing to internal GAPDH level.
Permix ex taq
Manufactured by Takara Bio
Sourced in China, Japan
Permix Ex Taq is a high-fidelity DNA polymerase designed for accurate DNA amplification. It is a premixed solution containing the Ex Taq DNA polymerase, buffer, and dNTPs, providing a convenient and ready-to-use formulation for PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green real time pcr master mix, by Takara Bio (3 mentions)
Primescript rt reagent kit, by Takara Bio (3 mentions)
Primerscript rt kit, by Takara Bio (1 mentions)
Abi prism 7500 sequence detection system, by Bio-Rad (1 mentions)
Mirvana mirna isolation kit, by Takara Bio (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Rna extraction kit, by Thermo Fisher Scientific (1 mentions)
Superscript 4 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Improm 2 reverse transcription system, by Promega (1 mentions)
Abi 7500ht real time pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
5 protocols using permix ex taq
1
Liver Tissue RNA Extraction and Gene Expression Analysis
2
Quantifying miR-200a Expression in Liver Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
miR-200a was isolated from cells or homogenized liver tissues using miRVana miRNA isolation kit (Takara, Dalian, China). Real time PCR assays were performed using PrimeScriptTM RT reagent Kits (TaKaRa), SYBR Green® miRcute miRNA Realtime PCR Kit (Tiangen, Beijing, China), SYBR Green® Realtime PCR Master Mix and Permix Ex Taq (TaKaRa) according to the manufacturer’s instructions.
Primers used were as follows: rat β-catenin, (Forward) 5′-CTT ACG GCA ATC AGG AAA GC-3′ and (Reverse) 5′-GAC AGA CAG CAC CTT CAG C-3′; GAPDH, (Forward) 5′-CGG ATT TGG TCG TAT TG-3′ and (Reverse) 5′-GAA GAT GGT GAT GGG ATT-3′. Primers for U6 and miR-200a were purchased from Sangon Biotech (Shanghai, China). The relative gene expression was normalized to the level of GAPDH while expression of miR-200a was normalized to the level of U6. All reactions were performed in triplicates for each sample. At least three independent experiments were carried out for each experimental condition.
Primers used were as follows: rat β-catenin, (Forward) 5′-CTT ACG GCA ATC AGG AAA GC-3′ and (Reverse) 5′-GAC AGA CAG CAC CTT CAG C-3′; GAPDH, (Forward) 5′-CGG ATT TGG TCG TAT TG-3′ and (Reverse) 5′-GAA GAT GGT GAT GGG ATT-3′. Primers for U6 and miR-200a were purchased from Sangon Biotech (Shanghai, China). The relative gene expression was normalized to the level of GAPDH while expression of miR-200a was normalized to the level of U6. All reactions were performed in triplicates for each sample. At least three independent experiments were carried out for each experimental condition.
3
Quantitative PCR Analysis of PRKACA and miR-503
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RT-qPCR assays for protein kinase CAMP-activated catalytic subunit alpha (PRKACA) and miR-503 expression were performed using a PrimeScript RT reagent kit, SYBR-Green Real-time PCR Master Mix and Permix Ex Taq (both from Takara, Dalian, China), and a TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA), respectively, according to the manufacturers' instructions. The parameters for PCR were as follows: Incubation of the templates at 94°C for 2 min, followed by 40 cycles of 94°C for 20 sec, 60°C for 1 min and 72°C for 20 sec, and finally incubation at 72°C for 2 min. We used U6 and GAPDH as the housekeeping genes for calculating the relative expression of miR-503 and PRKACA mRNA, respectively. The primers used were as follows: U6 forward, 5′-GCGCGTCGTGAAGCGTTC-3′ and reverse, 5′-GTGCAGGGTCCGAGGT-3′; PRKACA forward, 5′-GAGCAGGAGAGCGTGAAAGA-3′ and reverse, 5′-AGATCTGGATGGGCTGGTCT-3′; GAPDH forward, 5′-CGGAGTCAACGGATTTGGTCGTAT-3′ and reverse, 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′. The 2−ΔΔCq method (25 (link)) was used to evaluate the relative expression levels of the indicated genes.
4
Quantifying Molecular Regulators in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from cells by using RNA extraction kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. 2 µg RNA was synthesized into cDNA using SuperScriptTM IV First-Strand Synthesis System (Invitrogen, USA). Real-time qPCR of the reverse transcription products of TUG1, miR-194 and SIRT1 expression was determined using Permix Ex Taq (Takara, Tokyo, Japan), analyzed through the 7500 Real-time PCR System (Applied Biosystems, USA). The data were quantified by normalizing to GAPDH or U6. Relative expression level of genes was quantified using 2−△△Ct method. The primer sequences for qPCR were as follows:
miR-194 F: 5’-TGTAACAGCAACTCCATGTG-3’,
miR-194 R: 5’-GTCGTATCGAGAGCAGGGTCCGAGGTATTCGCACTCGATAC
GACTCCACAT-3’,
SIRT1 F: 5’-CAAACTTTGCTGTAACCCTGT-3’,
SIRT1 R: 5’-CAGCCACTGAAGTTCTTTCAT-3’,
TUG1 F: 5’-TAGCAGTTCCCCAATCCTTG-3’,
TUG1 R: 5’-CACAAATTCCCATCATTCCC-3’,
GAPDH F: 5’-AGGTCGGTGTGAACGGATTTG-3’,
GAPDH R: 5’- GGGGTCGTTGATGGCAACA-3’,
U6 F: 5’-CTCGCTTCGGCAGCACAT-3’,
U6 R: 5’-AACGCTTCACGAATTTGCGT-3’.
miR-194 F: 5’-TGTAACAGCAACTCCATGTG-3’,
miR-194 R: 5’-GTCGTATCGAGAGCAGGGTCCGAGGTATTCGCACTCGATAC
GACTCCACAT-3’,
SIRT1 F: 5’-CAAACTTTGCTGTAACCCTGT-3’,
SIRT1 R: 5’-CAGCCACTGAAGTTCTTTCAT-3’,
TUG1 F: 5’-TAGCAGTTCCCCAATCCTTG-3’,
TUG1 R: 5’-CACAAATTCCCATCATTCCC-3’,
GAPDH F: 5’-AGGTCGGTGTGAACGGATTTG-3’,
GAPDH R: 5’- GGGGTCGTTGATGGCAACA-3’,
U6 F: 5’-CTCGCTTCGGCAGCACAT-3’,
U6 R: 5’-AACGCTTCACGAATTTGCGT-3’.
5
Quantitative PCR analysis of gene expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
qPCR assay were performed as described before [29 (link)]. Briefly, total RNA was extracted using Trizol® (Invitrogen, USA) after cell treatment. ImProm-II Reverse Transcription System (Promega, USA) was used to generate First-strand cDNA. Real-time qPCR of the reverse transcription products were determined using Permix Ex Taq (Takara, Japan) and gene-specific primers were used for qPCR in an ABI 7500HT real time PCR system (Applied Biosystems, USA). The relative expression levels of RNAs were normalized with GAPDH or U6 and calculated using the comparative 2-ΔΔCT method. All experiments were performed at least three times. The primers used for qPCR were list as follow:
GAPDH-Forward: 5′-AGCCCAAGATGCCCTTCAGT-3′,
GAPDH-Reverse: 5′-CCGTGTTCCTACCCCCAATG-3′;
H19-Forward: 5′-AAGAGCTCGGACTGGAGACT-3′,
H19-Reverse: 5′-AAGAAGGCTGGATGACTGCC-3′;
miR-185-5p-Forward: 5′-CGCTGGAGAGAAAGGCAGT-3′,
miR-185-5p-Reverse: 5′-GTGCAGGGTCCGAGGT-3′;
U6-Forward: 5′-CTCGCTTCGGCAGCACA-3′,
U6-Reverse: 5′-AACGCTTCACGAATTTGCGT-3′;
IGF1-Forward: 5′-CTCTTCTACCTGGCGCTCTG-3′,
IGF1-Reverse: 5′-GCAACACTCATCCACAATGC-3′;
Osteocalcin (OCN)-Forward: 5′-AAGCAGGAGGGCAATAAGGT-3′,
OCN-Reverse: 5′-TAGGCGGTCTTCAAGCCATA-3′;
ALP-Forward: 5′-AACCCAGACACAAGCATTCC-3′,
ALP-Reverse: 5′-CCAGCAAGAAGAAGCCTTTG-3′;
Collagen I-Forward: 5′-CCCAGCCGCAAAGAGTCTAC-3′,
Collagen I-Reverse: 5′-AGCATACCTCGGGTTTCCAC-3′.
GAPDH-Forward: 5′-AGCCCAAGATGCCCTTCAGT-3′,
GAPDH-Reverse: 5′-CCGTGTTCCTACCCCCAATG-3′;
H19-Forward: 5′-AAGAGCTCGGACTGGAGACT-3′,
H19-Reverse: 5′-AAGAAGGCTGGATGACTGCC-3′;
miR-185-5p-Forward: 5′-CGCTGGAGAGAAAGGCAGT-3′,
miR-185-5p-Reverse: 5′-GTGCAGGGTCCGAGGT-3′;
U6-Forward: 5′-CTCGCTTCGGCAGCACA-3′,
U6-Reverse: 5′-AACGCTTCACGAATTTGCGT-3′;
IGF1-Forward: 5′-CTCTTCTACCTGGCGCTCTG-3′,
IGF1-Reverse: 5′-GCAACACTCATCCACAATGC-3′;
Osteocalcin (OCN)-Forward: 5′-AAGCAGGAGGGCAATAAGGT-3′,
OCN-Reverse: 5′-TAGGCGGTCTTCAAGCCATA-3′;
ALP-Forward: 5′-AACCCAGACACAAGCATTCC-3′,
ALP-Reverse: 5′-CCAGCAAGAAGAAGCCTTTG-3′;
Collagen I-Forward: 5′-CCCAGCCGCAAAGAGTCTAC-3′,
Collagen I-Reverse: 5′-AGCATACCTCGGGTTTCCAC-3′.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!