For qRT-PCR, total RNA was extracted from the hippocampus of approximately 9-month-old SARM1f/f, SARM1Nestin-CKO, APP/PS1;SARM1f/f and APP/ PS1;SARM1Nestin-CKO mice at using TRIzolTM Reagent (#15596026, Ambion), following the manufacturer’s instructions. The RNA was then converted into cDNA using the SuperScriptTM One-Step Reverse Transcription Kit (#10928-034, Invitrogen, CA, USA). RT-PCR detection system (Applied Biosystems, USA) was used to measure the expression levels of mRNA, and the iTaqTM Universal SYBR Green Supermix (Bio-Rad, USA) was used. Samples were independently amplified at least three times. The 2-ΔΔCt method was used to convert the relative gene expression against β-actin. The β-actin primer sequences were as follows: forward, 5'-AAGGAAGGCTGGAAAAGAGC-3' and reverse, 5'-GCTACAGCTTCACCACCACA-3'. TNF-α primer sequences were as follows: forward, 5'-ACGTGGAACT GGCAGAAGAG-3' and reverse, 5'-CTCCTCCACTT GGTGGTTTG-3' [36 (link)].
Rt pcr detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The RT-PCR detection system is a laboratory instrument designed for performing real-time reverse transcription polymerase chain reaction (RT-PCR) analysis. It is used to detect and quantify specific RNA sequences in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Itaq universal sybr green supermix, by Bio-Rad (1 mentions)
Sybr select master mix, by Thermo Fisher Scientific (1 mentions)
Reverse transcription kit, by Promega (1 mentions)
Trizol reagent, by Solarbio (1 mentions)
Primer express software, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using rt pcr detection system
1
Quantitative RT-PCR Analysis of Hippocampal Gene Expression
2
Quantitative Real-Time PCR Analysis of Cytokines
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Total RNAs in glomeruli and mesangial cells were extracted with TRIzol reagent (Solarbio, Beijing, China) strictly in line with specific instructions. Thereafter, 3 µg total RNA samples were collected for the synthesis of cDNA template by reverse transcription using a reverse transcription kit (Promega, Madison, WI, USA). qRT-PCR was carried out using SYBR Select Master Mix (Life Technologies, California, USA) and an RT-PCR detection system (Applied Biosystems, Foster City, CA, USA). The reaction procedure was as follows: 40 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 60 s. The following primers were used, for IFN-γ, sense: 5'-ACAACCCACAGATCCAGC-3', antisense: 5'-TCAGCACCGACTCCTTTT-3'; for TNF-α, sense: 5'-CCACGCTCTTCTGTCTACTG-3', antisense: 5'-GGGAACTTCTCCTCCTTGTT-3'; for IL-6, sense: 5'-GGAGTTCCGTTTCTACCT-3', antisense: 5'-CTCTGGCTTTGTCTTTCT-3'; for Ntrk1, sense: 5′-GGACAACCCTTTCGAGTTCA-3′, antisense: 5′-GTGGTGAACACAGGCATCAC-3′; for β-actin (control), sense: 5′-AGCGAGCATCCCCCAAAGTT-3′, antisense: 5′-GGGCACGAAGGCTCATCATT-3′. The relative expression of the other mRNAs was calculated by the 2−ΔΔCt method. The experiment was independently repeated three times.
3
Uterine Adiponectin Pathway Characterization
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Total RNAs were extracted from uterine tissue with Trizol Reagent (Invitrogen, CA, USA), according to the manufacturer's protocol. The RNAs were converted to cDNA with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). Real-time polymerase chain reaction (RT-PCR) was carried out with SYRB Select Master Mix (Applied Biosystems, USA) on a RT-PCR detection system (Applied Biosystems, USA). Reactions were performed as follows: incubation at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of amplification, each consisting of 95°C for 15 s, 60°C for 1 min, and then the temperature increased from 60°C to 95°C to obtain a melting curve. Primers for amplification of adiponectin (ADIPOQ), adiponectin receptors (ADIPOR1 and ADIPOR2), and the reference gene β-actin were designed using Primer Express Software (Applied Biosystems, USA). Primers were as follows: ADIPOQ, forward 5’-GGCCGTGATGGCAGAGAT-3’, reverse 5’-TTCCGCTCCTGTCATTCCA-3’; ADIPOR1, forward 5’-TTCCGCATCCACACAGAAAC-3’, reverse 5’-GCATGGTCAAGATTCCCAGAA-3’; ADIPOR2 forward 5’-TACACACAGAGACGGGCAACA-3’, reverse 5’-CCCCAGGCACAGGAAGAATA-3’; β-actin forward 5’-TCAGGTCATCACTATCGGCAAT-3’, reverse 5’-CATGGATGCCACAGGATTCC-3’. RT relative mRNA levels were calculated according to the relative quantification method (2−ΔΔCt) and expressed as the fold change relative to the control.
4
Liver Tissue RNA Expression Analysis
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TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) was used for extracting total RNA from liver tissue for spectro-photometrical quantification. RevertAid First Strand cDNA Synthesis Kit and oligo-dT primers (Thermo Fisher Scientific Ins, Burlington, ON, Canada) were used for reverse transcription of RNA into cDNA. For the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), hepatocyte growth factor (HGF) and heat shock protein 70 (HSP70)18 (link), specific primers were designed and rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as endogenous control. RT-PCR detection system (Applied Biosystems AB, USA) was used for quantitative real-time PCR amplification with a SYBR PCR Kit (TaKaRa Bio, Inc, Dalian, China). PCR amplification was performed with the following conditions: 1 cycle of 95 °C for 30 s; 40 cycles of 95 °C for 5 s, 55 °C (for GAPDH, HGF and HSP70) or 58 °C (for TNF-α and IL-6) for 15 s, and 72 °C for 15 s. 2−ΔΔCt method22 (link) was used for calculating relative mRNA expression. The results represent an x-fold induction versus the baseline levels in the PVL group.
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