Total cellular RNA was extracted, as previously described.29 (link) qRT-RT-PCR reactions containing cDNA, Sybr Green PCR Master Mix (Life Technologies) and primers for WIP1, Gli1, Ptc1, Trp53, and/or Glyceraldehyde-3 Phosphate Dehydrogenase (Gapdh) were run on an ABI 7500 Real-Time PCR Cycler (Life Technologies) using absolute quantification with a standard curve. Primer sequences available upon request. Absolute gene expression was determined based on standard curves. Target gene expression was normalized to Gapdh expression.
Abi 7500 real time pcr cycler
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI 7500 real time PCR cycler is a laboratory instrument designed for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. It is capable of performing real-time monitoring of DNA amplification during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Syber green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Abi 7500 software v2, by Thermo Fisher Scientific (1 mentions)
Platinum sybr green qpcr supermix udg, by Thermo Fisher Scientific (1 mentions)
Sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Gotaq rt qpcr system, by Promega (1 mentions)
Goscript reverse transcriptase kit, by Promega (1 mentions)
Small animal stereotaxic frame, by Kopf Instruments (1 mentions)
34 g beveled needles, by World Precision Instruments (1 mentions)
9 protocols using abi 7500 real time pcr cycler
1
Quantitative RT-PCR Analysis of Key Signaling Genes
2
Quantitative Real-Time RT-PCR Analysis
3
Quantitative RT-PCR Analysis of Key Signaling Genes
4
Quantitative Real-Time RT-PCR Analysis
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Total cellular RNA was extracted, as previously described.27 (link) Quantitative real-time, RT-PCR reactions containing cDNA, Syber Green PCR Master Mix (Life Technologies, Grand Island, NY) and primers for human WIP1, CXCR4, GRK5, and/or Glyceraldehyde-3 Phosphate Dehydrogenase (GAPDH) were run on an ABI 7500 Real-Time PCR Cycler (Life Technologies), using absolute quantification with a standard curve. Primer sequences are available upon request. Amplification products were verified by analysis of melting curves. Serial cDNA dilutions were used to determine standard curves for each primer. Analysis of housekeeping gene expression was included with each run. Absolute gene expression was determined based on standard curves. Target gene expression was normalized to GAPDH expression.
5
Quantitative Real-Time PCR from Cell Culture
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RNA extraction from cell culture and cDNA synthesis was performed as reported previously [35 (link)]. Quantitative real-time PCR was conducted using an ABI 7500 Real Time PCR cycler (Applied Biosystems) using ABI 7500 Software v2.3 (Applied Biosystems) with Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). The quantitative real-time PCR conditions were as described previously [35 (link)]. Quantitative real-time PCR Primers were purchased from Qiagen and Invitrogen (see supplementary Table 2 ). The ΔΔCt method was used to assess relative quantification. ACTB was used as control gene.
6
Quantitative Real-Time PCR Analysis
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Total RNA was purified using the RNeasy kit (Qiagen). Reverse transcription was performed with SuperScript III Reverse Transcriptase (Life Technologies) using oligo(dT) primers. Quantitative real-time PCR was carried out using the SYBR Green Master Mix (Applied Biosystems) in an ABI 7500 Real-Time PCR Cycler (Applied Biosystems). Results were calculated using the 2−ΔΔCt method; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal control. Primer sequences are listed in the Supplementary Table 2 .
7
Validation of RNA-seq Results by RT-qPCR
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Five genes were tested by RT-qPCR to validate RNA seq results. Gene candidates (listed in Table 1 ) satisfied the following criteria: (i) they were differentially expressed compared with the control condition, (ii) they were specific to a single condition, and (iii) they served an interesting putative biological function. For RT-qPCR analysis, cDNA was first synthesized from RNA samples by reverse transcriptase reaction using the GoScript® reverse transcriptase Kit (Promega, United States). RT-qPCR reactions were done in an ABI 7500 Real-Time PCR cycler (Applied Biosystems, Foster City, United States) using GoTaq RT-qPCR systems (Promega, United States) in a final volume of 10 μL. Primers were designed by using Primer Blast (Ye et al., 2012 (link); Optimal Tm = 62°C, 50–60% GC) and were used at a concentration of 0.5 μM. The cycling program consisted of 95°C for 5 min (initial denaturation); 40 cycles of 15 s at 95°C (denaturation) followed by 45 s at 62°C (annealing and extension); and an additional melting analysis of 40 min from 60 to 95°C. The ABI SDS software v.1.4 (Applied Biosystems, Foster City, United States) was used to collect the amplification data. Gene expression was calculated by using the 2–ddCt method (Zhang et al., 2020 ).
8
AAV-Mediated HA-FAAH Gene Delivery
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The N-terminally hemagglutinin (HA)-tagged mouse FAAH cDNA with 4 additional amino acids (Glu-Phe-Asp-Asn) between HA and the first codon of FAAH was cloned into the adeno-associated virus (AAV) plasmid containing the cytomegalovirus enhancer/chicken ß-actin (CAG) promoter [20 (link)], the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and the bovine growth hormone polyadenylation sequence (pA) flanked by AAV2 inverted terminal repeats. A transcriptional Stop cassette flanked by loxP sites was inserted downstream the CAG promoter to allow Cre recombinase-dependent transgene expression of HA-FAAH after injection into a Cre-expressing transgenic mouse line. Chimeric AAV serotype 1/2 vectors were produced and genomic titers determined using the Applied Biosystems ABI 7500 real time PCR cycler as described before [22 (link)].
9
AAV-Mediated Hippocampal CRIP1a Expression
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The rat Cnrip1a open reading frame was EcoRI and HindIII-linkered and fused downstream of the hemagglutinin (HA) epitope tag coding region in an AAV expression cassette containing the 1.1 kb CMV immediate early enhancer/chicken βactin hybrid promoter (CAG), the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and the bovine growth hormone polyadenylation sequence (bGHpA) flanked by AAV2 inverted terminal repeats (pAAV-CRIP1a). The same backbone carrying no cDNA was used as control (pAAV-empty). Production of neurotropic AAV1/2 vectors and determination of genomic titres using the ABI 7500 real time PCR cycler (Applied Biosystems) were performed as described (During et al. 2003) . Adult male C57BL/6N mice were anaesthetized by intraperitoneal injection of fentanyl (0.05 mg/kg), midazolam (5 mg/kg) and medetomidin (0.5 mg/kg), and fixed in a small animal stereotaxic frame (Kopf instruments, Tujunga, CA). One microliter of either AAV-CRIP1a or AAV-empty (9×10 10 viral genomes/ml) was injected bilaterally into the CA1 area of the dorsal hippocampus (+2.0 mm AP, ±1.6 mm ML, -1.3 mm DV from bregma) at a rate of 150 nl/min using a microprocessorcontrolled mini-pump with 34G beveled needles (World Precision Instruments, Sarasota, FA).
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