Total RNA was extracted from forebrain samples with TRIzol reagent (Invitrogen). Reverse transcription was performed using SuperScript III reverse transcriptase reagents (Invitrogen). Quantitative PCR was done using Perfecta SYBR Green Fastmix (Quanta Biosciences) utilizing the ABI Prism 7000 detection system (Applied Biosystems). For expression studies, the qRT-PCR results were normalized against an internal control (GAPDH). Primers were designed with Primer Express Version 2.0 software (Applied Biosystems) using sequence data from NCBI. GAPDH primers were used as an internal control for each specific gene amplification. The relative levels of expression were quantified and analyzed by using ABI PRISM Sequence Detection System 7000 software. The real-time value for each sample was averaged and compared using the comparative CT method. The relative amount of target RNA was calculated relative to the expression of endogenous reference and relative to a calibrator which was the mean CT of control samples.
Abi prism 7000 detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI Prism 7000 detection system is a real-time PCR instrument designed for quantitative analysis of nucleic acids. It features a 96-well thermal cycler and utilizes fluorescence detection to monitor amplification of DNA or RNA targets during the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green pcr master mix kit, by Thermo Fisher Scientific (4 mentions)
Primer express software v2, by Thermo Fisher Scientific (4 mentions)
Abi prism 7000 sds software, by Thermo Fisher Scientific (3 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Superscript 3 reverse transcriptase reagent, by Thermo Fisher Scientific (1 mentions)
Primer express version 2, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Perfecta sybr green fastmix, by Quanta Biosciences (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (1 mentions)
Absolute qpcr sybr green rox mix, by Thermo Fisher Scientific (1 mentions)
Quick rna microprep kit, by Zymo Research (1 mentions)
Taqman mirna probes, by Thermo Fisher Scientific (1 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Taqman universal master mix 2, by Thermo Fisher Scientific (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Amplitaq gold pcr master mix, by Thermo Fisher Scientific (1 mentions)
Kod sybr qpcr mix, by Toyobo (1 mentions)
13 protocols using abi prism 7000 detection system
1
Forebrain Total RNA Extraction and qRT-PCR
2
Hypoxia-Induced Gene Expression in MSCs
3
Quantification of Gene Silencing by qRT-PCR
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qRT-PCR was performed with the above mentioned extracted total RNAs to quantify the level of gene silencing. Also RNA extracted from the GS-tub-GAL4 line was tested by qRT-PCR to identify possible leakiness of the tubulin driver. PCR reactions were performed in MicroAmp optical 96-well reaction plates using a SYBR-Green PCR master mix kit (Applied Biosystems), according to the manufacturer's instructions. Primers for qRT-PCR were designed using Primer Express Software v2.0 (Applied Biosystems). The forward primer used in the reaction for β-CA gene expression was 5’-GACAAGGGAGCAAATGGTCAA-3’ and the reverse primer was 5’-TCTACTGTCCATGCAGGTGAAGAA-3’. The β-CA amplicon size was 88 bp. The reaction was carried out in an ABI PRISM 7000 Detection System (Applied Biosystems). The data were analyzed with ABI PRISM 7000 SDS software and normalized to the RpL32 housekeeping gene. The forward primer used for RpL32 was 5’-GGTTACGGATCGAACAAGCG-3’ and the reverse primer was 5’-TTCTGCATGAGCAGGACCTC-3’. The RpL32 amplicon size was 101 bp. The final results were expressed as % of expression of the control lines, which were normalized to 100 % each, as described in [26 (link)].
4
RNA Quantification by RT-qPCR
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Total RNA were reverse transcribed in 20 μl using the High Capacity cDNA Reverse Transcription Kit, according to the manufacturer's protocol (Applied Biosystem). Quantitative real-time PCR was performed using 80 ng of cDNA in a final volume of 20 μl according to the manufacturer's instructions (Applied Biosystems), on the ABI Prism 7000 Detection System (Applied Biosystems). Relative expression was calculated using the comparative Ct method. All PCRs were performed in triplicate including no-template controls.
5
Quantitative PCR Analysis of Alveolar Macrophages
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Total RNA was extracted from alveolar macrophages using Quick-RNA MicroPrep kit (ZYMO, Freiburg, Germany). 50–1000 ng total RNA was used for cDNA synthesis by Superscript II Reverse Transcriptase kit with the protocol described previously.63 (link) To determine the expression of target genes relative to the actin housekeeper Actb, the ABsolute QPCR SYBR Green ROX Mix (Thermo Scientific, Wilmington, DE, USA) was used on an ABI PRISM 7000 detection system (Applied Biosystems, Foster City, CA, USA). Primer sequences are given in Supplementary Table I . Relative expression of target genes and housekeeping gene Actb was calculated according to the 2−ΔCt method.63 (link)
6
Quantifying Gene Expression in HUVECs
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To evaluate the effect of the tested drugs on nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, superoxide dismutase (SOD), endothelial and inducible nitric oxide synthase (eNOS and iNOS) expression at the level of messenger RNA (mRNA), HUVECs were grown on 24-well microplates. mRNA levels were determined by real-time qPCR as previously described.19 ,20 (link) Briefly, total RNA was extracted from the cells using Trizol reagent and was processed directly to complementary DNA (cDNA) synthesis using the TaqMan Reverse Transcription Reagents kit according to the manufacturer’s protocol. cDNA was subsequently amplified by MasterAmp Pfl DNA polymerase using the ABI Prism 7000 detection system (Applied Biosystems, USA). Specific primer sequences were designed based on primary mRNA sequences from GenBank. β-actin was used as an active and endogenous reference to correct for differences in the amount of total RNA added to the reaction and to compensate for different levels of inhibition during RT of RNA and during PCR. To compensate for variations in input RNA amounts and efficiency of RT, β-actin mRNA was quantified and the results were normalized to these values. Relative mRNA expression was calculated using the formula: 2−∆∆Ct (2−(Cttarget gene – Ctreference gene)), where Ct is a copy threshold.
7
Quantitative PCR Primer Design and Gene Expression Analysis
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Real‐time quantitative PCR (qPCR) primers were designed based on the transcript sequences taken from Ensembl (www.ensembl.org ; ENST00000317995, ENST00000084798, ENST00000285273, and ENST00000369626 for CA8, CA10, CA11, and SLC16A1, respectively), using the program Primer Express® Software v2.0 (Applied Biosystems). Real‐time qPCR was performed using the SYBR Green PCR Master Mix Kit in an ABI PRISM 7000 Detection System™ according to the manufacturer's instructions (Applied Biosystems). The PCR conditions consisted of an initial denaturation step at 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s (denaturation) and 60 °C for 1 min (elongation). The data were analyzed using the ABI PRISM 7000 SDS™ software (Applied Biosystems). Every PCR was performed in a total reaction volume of 15 μL containing 2 μL of first‐strand cDNA (20 ng cDNA), 1 × Power SYBR green PCR Master Mix™ (Applied Biosystems), and 0.5 μm of each primer. The final results expressed as the N‐fold relative difference (ratio) in gene expression between the studied samples. The relative expression values were calculated according to the equation of Pfaffl with appropriate modification 44 .
8
Quantitative RT-PCR Analysis of miRNA Expression
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Samples to be used for miRNA analysis were isolated using the miRVana miRNA isolation kit (Applied Biosystems) as described above.cDNA was synthesised from 10 ng RNA/RT reaction with miRNA‐specific primers for TaqMan assays (Applied Biosystems) using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) as manufacturer's instructions.qRT‐PCR was carried out using TaqMan Universal Master Mix II (Applied Biosystems) with Taqman miRNA probes (Applied Biosystems) according to manufacturer's instructions. All reactions were performed in quadruplicate per sample on an ABI Prism 7000 detection system (Applied Biosystems) with thermal cycling at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 sec and 60 °C for 1 min. Gene expression levels were normalised to U6 snRNA using the 2‐ΔΔCt method. Preliminary experiments had shown that U6 snRNA was a suitable control for this purpose (data not shown). Assays used were: hsa‐miR‐19b, hsa‐miR‐20b, hsa‐miR‐363#, hsa‐miR‐363, hsa‐miR‐18b, hsa‐let‐7a, hsa‐let‐7f, custom xtr‐mir‐106a (Applied Biosystems). It is important to note that the inclusion of stem loop structures in the primers used in the miRNA specific cDNA syntheses allow for the detection of mature, biologically active miRNAs.
9
Real-Time Quantitative PCR Protocol
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Real time quantitative PCR (RT-qPCR) primers were designed based on the complete cDNA sequences taken from Ensembl (Transcript IDs as above: using the program Primer Express Software v2.0 (Applied Biosystems)) (Table 1 ). The RT-qPCR was performed using a SYBR Green PCR Master Mix Kit in an ABI PRISM 7000 Detection System according to the manufacturer's instructions (Applied Biosystems). The PCR conditions consisted of an initial denaturation step at 95°C for 10 min followed by 40 cycles at 95°C for 15 sec (denaturation) and 60°C for 1 min (elongation). The data was analyzed using the ABI PRISM 7000 SDS software (Applied Biosystems). Each PCR reaction was performed in a total reaction volume of 15 μl containing 20ng of cDNA, 1 × Power SYBR green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), and 0.5 μM of each primer. The relative expression of the genes between the studied samples, and internal control gene β-actin were calculated according to Pfaffl's equation [34 (link)].
10
Quantitative Real-Time PCR for Gene Expression Analysis
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Quantitative real-time PCR (qRT-PCR) primers were designed based on the complete cDNA sequence taken from Ensembl (ENSDART00000057097), using the program Primer Express® Software v2.0 (Applied Biosystems) (forward primer 5′-CAAACATTTATTTGCCAGCACTCC-3′ and reverse primer 5′-TATGTCCAATAATCTCCATCTACTCC-3′). qRT-PCR was performed using the SYBR Green PCR Master Mix Kit in an ABI PRISM 7000 Detection System™ according to the manufacturer’s instructions (Applied Biosystems). The PCR conditions consisted of an initial denaturation step at 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s (denaturation) and 60 °C for 1 min (elongation). The data were analyzed using the ABI PRISM 7000 SDS™ software (Applied Biosystems). Every PCR was performed in a total reaction volume of 15 μl containing 2 μl of first strand cDNA (20 ng cDNA), 1 × Power SYBR green PCR Master Mix™ (Applied Biosystems, Foster City, CA, USA), and 0.5 μM of each primer. We performed these experiments in duplicate and with sample duplicates. The results of ca6 gene expression were normalized using zebrafish housekeeping gene gapdh as internal control. The final results are given as relative expression values, calculated according to the Pfaffl’s equation (Pfaffl, 2001 (link)).
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