Total RNA was isolated with Trizol Reagent (Invitrogen) according to the manufacturer’s instruction and reversely transcribed into cDNA using M-MLV Reverse Transcriptase (Takara, Japan) and oligo (dT)18 primer (Takara, Dalian, China). The following specific primers were used in RT-qPCR of human lncRNA-CR594175 and β-actin: lncRNA-CR594175: forward 5′-TTATGACACATGCCACAACA-3′ and reverse 5′-GGTACCTGTTATAAGTAGAATCA-3′; β-actin: forward5’-CCTGTACGCCAACACAGTGC-3′ and reverse 5′-ATACTCCTGCTTGCTGATCC-3′. The lengths of amplified products were 109 bp and 211 bp, respectively. RT-qPCR was performed using SYBR Premix Ex Taq kit and TP800 System (Takara). cDNA from 200 ng total RNA was used as the template. The PCR reaction was carried out under the following conditions: 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 20 s and extension at 72 °C for 20 s. The relative levels of mRNA and hsa-miR-142-3p were normalized using the 2-△△Ct method by using β-actin and U6 as the references. The PCR primers for hsa-miR142-3p or U6 were as follows: hsa-miR142-3p: forward: 5′- TGTAGTGTTTCCTACTTTATGGA-3′ and reverse: 5′-GTCGTATCCAGTGCGTGTCGTG-3′; U6: forward:5′-GTGCTCGCTTCGGCAGCACAT-3′ and reverse: 5′-TACCTTGCGAAGTGCTTAAAC-3′.
Tp800 system
Manufactured by Takara Bio
Sourced in China
The TP800 System is a laboratory instrument designed for automated sample preparation and purification. It utilizes a compact benchtop design and can handle a range of sample types and volumes. The system is capable of performing various purification protocols, including nucleic acid extraction and protein purification.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (10 mentions)
Sybr premix ex taq kit, by Takara Bio (6 mentions)
M mlv reverse transcriptase, by Takara Bio (5 mentions)
Oligo dt 18 primer, by Takara Bio (4 mentions)
Genelute mrna miniprep kit, by Merck Group (2 mentions)
Gotaq qpcr master mix, by Promega (2 mentions)
Uv 1700, by Shimadzu (2 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Sybr premix ex taq tli rnaseh plus, by Takara Bio (1 mentions)
Random primer, by Thermo Fisher Scientific (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Rnase free dnase, by Qiagen (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Sybr premix ex taqtm kit, by Takara Bio (1 mentions)
12 protocols using tp800 system
1
Quantification of lncRNA-CR594175 and miR-142-3p
2
Real-Time PCR Analysis of Gene Expression
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Total RNA was extracted from SACC-LM cells with RNAiso Plus (TRIzol; Takara Bio, Inc.), and then, reverse transcription into cDNA was performed using a PrimeScript RT reagent Kit (Takara Bio, Inc.) according to the manufacturer's instructions. The primer sequences for RT-qPCR are provided in Table SIII . GAPDH was used as the internal control for each experiment. RT-qPCR was performed with SYBR Premix Ex Taq (Tli RNaseH Plus; Takara Bio, Inc.). The PCR conditions were as follows: Initial denaturation at 95°C for 30 sec, followed by 40 cycles of denaturation at 95°C for 5 sec, annealing at 55°C for 30 sec and elongation at 72°C for 30 sec, and then a final extension at 72°C for 5 min. The data were collected with a TP800 system (Takara Bio, Inc.) and analyzed using the 2−ΔΔCq comparative method (26 (link)).
3
Somatostatin Receptor Expression Analysis
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Total RNA was isolated with the RNeasy Mini Kit (QIAGEN, Hilden, Germany) and treated with RNase-Free DNase (QIAGEN). cDNA was generated from DNase-treated total RNA by using random primers (Invitrogen, Carlsbad, CA) with Superscript II reverse transcriptase (Invitrogen). The primer sequences were as follows: SST forward, 5ʹ-CCA GAC TCC GTC AGT TTC TGC A-3ʹ; SST reverse, 5ʹ-CAT CAT TCT CCG TCT GTT TGG GTT-3ʹ [10 (link)]; SSTR1 forward, 5ʹ- TCT GCG CGA AGA TCG TCA AC-3ʹ; SSTR1 reverse, 5ʹ- GCG GCT CTG GAC TGG TAA ATG-3ʹ (TaKaRa, Tokyo, Japan); GAPDH forward, 5ʹ-GCA CCG TCA AGG CTG AGA AC-3ʹ; and GAPDH reverse, 5ʹ-TGG TGA AGA CGCCAG TGG A-3ʹ. To analyze the expression of SSTR2, SSTR3, SSTR4, and SSTR5, we used previously described primers and conditions. [19 (link)] Quantitative-RT-PCR was performed on the TaKaRa TP800 system. Quantitative RT-PCR was carried out as described previously. [4 (link)]
4
Quantitative Analysis of Smad4 Expression
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Total RNA was isolated with Trizol Reagent (Invitrogen) according to the manufacturer’s instruction and reversely transcribed into cDNA using M-MLV Reverse Transcriptase (Takara BIO, Japan) and oligo (dT)18 primer (Takara BIO). The following specific primers were used in the quantitative PCR of rat Smad4 and β-actin: Smad4: 5’- TGGACATTACTGGCCGGTTCACAA-3’ and 5’-GCCAAGCAAAAGCGATCTCCTCCAG -3’ and β-actin: 5’-CCTGTACGCCAACACAGTGC-3’ and 5’-ATACTCCTGCTTGCTGATCC-3’. The lengths of the amplified products were 219 bp and 211 bp, respectively. Real-time PCR was performed using a SYBR® Premix Ex Taq™ kit (Takara BIO) and a TP800 System (Takara BIO). cDNA from 200 ng total RNA was used as the template. The PCR reactions was carried out under the following conditions: 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 20 s and extension at 72°C for 20 s. The mRNA levels of Smad4 were normalized using the delta-delta Ct method to the expression of β-actin, an endogenous housekeeping gene.
5
Quantitative RT-PCR Analysis of iPSCs
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Total RNA was extracted from the differentiated iPSCs using TRIZOL reagent (Invitrogen) according to the manufacturer's instructions. After reverse transcription, quantitative real-time polymerase chain reaction (qRT-PCR) was performed using a TP800 system (Takara, Japan) with SYBR Premix Ex Taq (Takara). The qRT-PCR conditions were 40 cycles at 95°C for 10 s, 60°C for 20 s and 72°C for 20 s. GAPDH was used as the internal control. The primer sequences used are listed in Table 1 . Relative mRNA expression of cells cultured on scaffolds and plates was calculated.
6
Quantifying miR338-3p Expression by RT-qPCR
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In this study, RT-qPCR was mainly used to detect the relative contents of miR338-3p in sample of cells. Briefly, after 2 rounds of Dulbecco's Phosphate-Buffered Saline (dPBS) cleaning, approximately 1 × 106 cells were fully lysed by 1 ml Trizol Reagent (15596018, Thermofisher), followed by extracting total RNA using GenElute mRNA Miniprep Kit (RTN350. Sigma-Aldrich), and finally measuring its concentration using a UV fluorescence photometer (UV-1700, Shimadzu, Japan). Two micrograms RNA of each sample was used to produce cDNA with the 3′-specific primers, 5′-TACCTTGCGAAGTGCTTAAAC-3′ for human U6 snRNA and 5′-GTCGTATCC AGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGAACAACAA-3′ for miR338-3p. After that, quantitative-PCR was carried out using a GoTaq qPCR Master Mix (Promega Corporation) and the TP800 System (Takara Bio Inc.) with three repeated reactions. The differences between groups in expression levels of miR338-3p were analyzed using the 2−ΔCt method, and U6 snRNA was used as internal reference. The pairs of PCR primers were as follows: U6-forward 5′-GTGCTCGCTTCGGCAGCACAT-3′ and U6-reverse 5′-TACCTTGCGAAGTGCTTAAAC-3′; miR338-3p-forward 5′-GCCGGCGCCCGAGCTCTGGCTC-3′ and miR338-3p-reverse 5′-TCCAGCATCAGTGATTTTGTTG-3′.
7
Quantifying miR338-3p Expression by RT-qPCR
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In this study, RT-qPCR was mainly used to detect the relative contents of miR338-3p in sample of cells. Briefly, after 2 rounds of Dulbecco's Phosphate-Buffered Saline (dPBS) cleaning, approximately 1 × 106 cells were fully lysed by 1 ml Trizol Reagent (15596018, Thermofisher), followed by extracting total RNA using GenElute mRNA Miniprep Kit (RTN350. Sigma-Aldrich), and finally measuring its concentration using a UV fluorescence photometer (UV-1700, Shimadzu, Japan). Two micrograms RNA of each sample was used to produce cDNA with the 3′-specific primers, 5′-TACCTTGCGAAGTGCTTAAAC-3′ for human U6 snRNA and 5′-GTCGTATCC AGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGAACAACAA-3′ for miR338-3p. After that, quantitative-PCR was carried out using a GoTaq qPCR Master Mix (Promega Corporation) and the TP800 System (Takara Bio Inc.) with three repeated reactions. The differences between groups in expression levels of miR338-3p were analyzed using the 2−ΔCt method, and U6 snRNA was used as internal reference. The pairs of PCR primers were as follows: U6-forward 5′-GTGCTCGCTTCGGCAGCACAT-3′ and U6-reverse 5′-TACCTTGCGAAGTGCTTAAAC-3′; miR338-3p-forward 5′-GCCGGCGCCCGAGCTCTGGCTC-3′ and miR338-3p-reverse 5′-TCCAGCATCAGTGATTTTGTTG-3′.
8
Quantitative Analysis of Gene and miRNA Expression
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Total RNA was isolated from cells or tissues and reverse-transcribed into cDNA using M-MLV reverse transcriptase (Takara Bio). RT-qPCR was performed using the SYBR Premix Ex TaqTM kit and TP800 System (Takara Bio). The levels of IMAT1, KLF4, and Snai1 were calculated using β-actin as an internal control, and hsa-miR22-3p levels were normalized by U6 as a reference. The PCR primers used are provided in Supplementary Table S1 .
9
Quantification of Taurine Transporters
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Total RNA was isolated with Trizol Reagent (Invitrogen) according to the manufacturer’s instructions and reverse transcribed into cDNA using M-MLV Reverse Transcriptase and Random9 primer (Takara Bio, Kusatsu, Siga, Japan). The following specific primers were used for quantitative PCR of rat TauT (Forward: 5'-GAGCGGCCTGCCTGTGTTT-3', Reverse: 5'- CCCAGGCCAGGATGACGATGTAGT-3'), CDO (Forward: 5'-GAAGCCTACGAGAGCAATCCT-3', Reverse: 5'- TCACCCCAGCACAGAATCATC-3'), CSD (Forward: 5'-GGCCGGGCGCATCATTACG-3', Reverse: 5'-AAGACCCCATCCCCAGTGTTCC-3') and β-actin (Forward: 5'-TTCCAGTGACTCCACGTGC-3', Reverse: 5'- AACTTTGGGCCTGTGCCGAAGGGT-3'). The lengths of the amplified products were 167, 125, 138, and 215 bp, respectively. Real-time PCR was performed using SYBR Premix Ex Taq™ kit and the TP800 System (Takara). The cDNA from 200 ng total RNA was used as the template. The PCR reactions were performed under the following conditions: 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 20 s, and extension at 72 °C for 20 s. The TauT mRNA levels were normalized using the delta-delta-Ct method, to the expression of an endogenous housekeeping gene, β-actin.
10
Quantitative Analysis of CR594175 and miR-142-3p
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Total RNA was isolated with Trizol Reagent (Invitrogen) according to the manufacturer's instruction and reversely transcribed into cDNA using M-MLV Reverse Transcriptase (Takara, Japan) and oligo(dT)18 primer (Takara, Dalian, China). The following speci c primers were used in RT-qPCR of human CR594175 and β-actin: CR594175: forward 5'-TTATGACACATGCCACAACA-3' and reverse 5'-GGTACCTGTTATAAGTAGAATCA-3' ; and β-actin: forward5'-CCTGTACGCCAACACAGTGC-3' and reverse 5'-ATACTCCTGCTTGCTGATCC-3'. The lengths of ampli ed products were 109 bp and 211 bp, respectively. RT-qPCR was performed using SYBR Premix Ex Taq kit and TP800 System (Takara ,Dalian, China). cDNA from 200 ng total RNA was used as the template. The PCR reactions was carried out under the following conditions: 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 20 s and extension at 72°C for 20 s. The relative levels of mRNA and hsa-miR-142-3p were normalized using the 2 - △△Ct method by using β-actin and U6 as the references. The PCR primers for hsa-miR142-3p or U6 were as follows: hsa-miR142-3p sense: forward: 5′-TGTAGTGTTTCCTACTTTATGGA-3′ and reverse: 5′-GTCGTATCCAGTGCGTGTCGTG-3′; U6 sense: forward:5′-GTGCTCGCTTCGGCAGCACAT-3′ and reverse: 5′-TACCTTGCGAAGTGCTTAAAC-3′.
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