Total RNA extraction, reverse transcription and TaqMan real-time polymerase chain reaction (PCR) for miRNAs were performed according to the manufacturer's instructions as described previously [44 ]. To quantify SIRT1 mRNA, 1 μg of total RNA was reverse-transcribed to cDNA using oligo dT and Thermoscript (TaKaRa), performed using the following conditions: 42°C for 60 min and 85°C for 5 min. SYBER Green Dye (Invitrogen), and specific primers for SIRT1 and GAPDH were used. The sequences of the primers were as follows: SIRT1 (sense): 5′-CTGTTTCCTGTGGGATACCTGACT-3′; SIRT1 (antisense): 5′-ATCGAACATGGCTTGAGGATCT-3′; GAPDH (sense): 5′-GATATTGTTGCCATCAATGAC-3′; GAPDH (antisense): 5′-TTGATTTTGGAGGGATCTC G-3′. The relative amount of SIRT1 mRNA was normalized to GAPDH.
Thermoscript
Manufactured by Takara Bio
Sourced in China
Thermoscript is a laboratory equipment designed for precise temperature control and monitoring. It is a core tool used in various scientific applications that require accurate thermal management, such as PCR (Polymerase Chain Reaction) and other temperature-sensitive experiments.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Sybr green dye, by Thermo Fisher Scientific (2 mentions)
Abi 7300 sequence detection system, by Thermo Fisher Scientific (2 mentions)
Syber green dye, by Thermo Fisher Scientific (1 mentions)
Oligo dt, by Takara Bio (1 mentions)
Applied biosystems 7300 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Taqman mirna probes, by Thermo Fisher Scientific (1 mentions)
Sybr green dye, by Roche (1 mentions)
4 protocols using thermoscript
1
Quantifying SIRT1 mRNA Expression by RT-qPCR
2
Quantitative Gene and miRNA Expression Analysis
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Total RNA was extracted from tissues and the cultured cells using Trizol reagent (Invitrogen) according to the manufacturer's instructions. For quantitative RT-PCR analysis of mRNA, 1 μg of total RNA was reverse transcribed to cDNA with oligd(T) and Thermoscript (TaKaRa, Dalian, China). Real-time PCR was performed on an Applied Biosystems 7300 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using SYBR green dye (Roche, Mannheim, Germany). The sequences of the primers used for gene amplification were as follows: Apaf-1 (forward): 5′-GTTGATGCTGTCATTATGTAGGC-3′ Apaf-1 (reverse): 5′-AGGTAAAAGGGGAAGTATGTGTT-3′ β-actin (forward): 5′-CTGTCCCTGTATGCCTCTG-3′ and β-actin (reverse): 5′-ATGTCACGCACGATTTCC-3′. Quantitative RT-PCR of mature miRNAs was performed using TaqMan miRNA probes (Ambion, Austin, TX, USA) according to the manufacturer's instructions. β-Actin and U6 snRNA were used for normalization in gene and miRNA expression studies, respectively. A relative fold change in expression of the target gene transcript was calculated with the equation 2−ΔCT.
3
Quantification of TXNIP and p27 Expression
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Total RNA was extracted from cultured cells using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. For quantitative RT-PCR analysis of TXNIP and p27, 1μg total RNA was reverse transcribed to cDNA with oligdT primers and Thermoscript (TaKaRa, Dalian, China). Primer sequences (forward and reverse, respectively) were as follows: TXNIP, 5′-AGAGCCAACAGAACAGAAGAA-3′ and 5′-AGAGGCAGATCATTTAAGAGTG-3′; p27, 5′-AGAGCCAACAGAACAGAAGAA-3′ and 5′-AGAGGCAGATCATTTAAGAGTG-3′; β-actin, 5′-AGGGAAATCGTGCGTGAC-3′ and 5′-CGCTCATTGCCGATAGTG-3′. Real-time PCR analyses of TXNIP and p27 were performed on an ABI 7300 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using SYBR green dye (Invitrogen, Carlsbad, CA, USA). A 20 μl reaction volume included 1μl cDNA, 1× QuantiTect SYBR green PCR Master Mix, and 0.5 μM of sense and 0.5 μM of antisense primer. All PCRs were performed in triplicate. Threshold cycles (CT) were determined using fixed threshold settings.
4
Quantitative RT-PCR analysis of BRCA1, AR, and PARP1
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Total RNA was extracted from cultured cells using Trizol Reagent (Invitrogen, Carlsbad,CA,USA) according to the manufacturer's instructions. For quantitative RTPCR analysis of BRCA1, AR and PARP1, 2µg total RNA was reverse transcribed to cDNA with oligdT primers and Thermoscript (TaKaRa, Dalian, China). Primer sequences (forward and reverse, respectively) were as follows: BRCA1, 5'-CCAAGGTTAGAGAGTTGGACAC-3' and 5'-GAAACCGTGCCAAAAGACTTC-3'; AR, 5'-GGGCGAAGTAGAGCATCCT-3' and 5'-GACGACCAGATGGCTGTCATT-3'; PARP1, 5'-TTTCCATCAAACATGGGCGAC-3' and 5'-CGGAGTCTTCGGATAAGCTCT-3'; GAPDH, 5'-AAGTGGTCGTTGAGGGCAATG-3' and 5'-CTGGGCTACACTGAGCACC-3'. Real-time PCR analyses of BRCA1, AR and PARP1 were performed on an ABI 7300 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using SYBR green dye (Invitrogen, Carlsbad, CA, USA). A 20 μl reaction volume included 1μl cDNA, 1× QuantiTect SYBR green PCR Master Mix, and 0.5 μM of sense and 0.5 μM of antisense primer. All PCRs were performed in triplicate. Threshold cycles (CT) were determined using fixed threshold settings.
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