Real-time quantitative PCR was performed using ABI 7000 SDS with the Taqman probe mixture (Applied Biosystems). Probes/primes for the analysed genes related to β-endorphin messenger expression were designed with Primer Express 2.0 software. The oligonucleotide probes were tagged with the reporter dye 6-FAM at its 5’-end and the quencher TAMRA dye at the 3’-terminal side. The internal control was chosen as the housekeeping gene actin. The PCR reaction was performed with the routine protocol of 45 cycles. The relative expression levels of the gene were compared.
Abi 7000 sds
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ABI 7000 SDS is a real-time PCR system designed for gene expression analysis and quantification. It features a 96-well block format and provides thermal cycling, data collection, and analysis capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Taqman probe mixture, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Power sybr green, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
7000 system sds software v1, by Thermo Fisher Scientific (2 mentions)
Sybr green chemistry, by Thermo Fisher Scientific (1 mentions)
Primer express, by Thermo Fisher Scientific (1 mentions)
Rneasy plus universal mini kit, by Qiagen (1 mentions)
Quantus fluorometer, by Promega (1 mentions)
Random primer, by Promega (1 mentions)
9 protocols using abi 7000 sds
1
Quantitative Analysis of β-Endorphin Expression
2
Gene Expression Analysis in Drosophila
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Total RNA was prepared from 20 whole, age-matched flies of indicated genotype and treatment using Trizol (Invitrogen, Carlsbad, USA). At least three independent RNA extractions were prepared for each sample. Relative message abundance was determined by amplification and staining with SYBR Green I using an ABI 7000 SDS (Applied Biosystems). Expression of Rp49 was used for normalization. Differences between genotypes were assessed by t-test or nested ANOVA. Primer sequences are listed below.
Mthl3:
Forward: 5′-GATCCCCGCCCATTTGACAG-3′
Reverse: 5′-GGCTCGCCACCTTCTCCTTC-3′
Mthl9:
Forward: 5′-TACGCCCACACGGTCAACAT-3′
Reverse: 5′-GCCCGGTACTCCACTCCATC-3′
Mthl11:
Forward: 5′-GCAAGCGGTGGGTTTTCTGT-3′
Reverse 5′-TTCTACGTCGGCCATTTTCTCA-3′
Gr64a:
Forward: 5′-ACGGCGGCGGACATCAAT-3′
Reverse: 5′-CTCCACCTCGACGCACCAG-3′
Gr98a:
Forward: 5′-CATGCGGCGACTGATGAAGTGT-3′
Reverse: 5′-CGAAGCTGAAGCGCCAGTAGC-3′
Gr32a:
Forward: 5′-TCGCATCGGCTTTGCTCAGG-3′
Reverse: 5′-CGCCTCGCTCGTGCTCCAC-3′
Or59b:
Forward: 5′-GCCGGGCGAGTTCCTTACCT-3′
Reverse: 5′-CGTTCGCCAGCCTCTTGTCC-3′
Or83b:
Forward: 5′-CCAGAAAGAACAGCTTCCTCATCT-3′
Reverse: 5′-CGAGTCGGATGCTCGTTACC-3′
Rp49:
Forward: 5′-ACTCAATGGATACTGCCCAAGAA-3′
Reverse: 5′-CAAGGTGTCCCACTAATGCATAAT-3′
Mthl3:
Forward: 5′-GATCCCCGCCCATTTGACAG-3′
Reverse: 5′-GGCTCGCCACCTTCTCCTTC-3′
Mthl9:
Forward: 5′-TACGCCCACACGGTCAACAT-3′
Reverse: 5′-GCCCGGTACTCCACTCCATC-3′
Mthl11:
Forward: 5′-GCAAGCGGTGGGTTTTCTGT-3′
Reverse 5′-TTCTACGTCGGCCATTTTCTCA-3′
Gr64a:
Forward: 5′-ACGGCGGCGGACATCAAT-3′
Reverse: 5′-CTCCACCTCGACGCACCAG-3′
Gr98a:
Forward: 5′-CATGCGGCGACTGATGAAGTGT-3′
Reverse: 5′-CGAAGCTGAAGCGCCAGTAGC-3′
Gr32a:
Forward: 5′-TCGCATCGGCTTTGCTCAGG-3′
Reverse: 5′-CGCCTCGCTCGTGCTCCAC-3′
Or59b:
Forward: 5′-GCCGGGCGAGTTCCTTACCT-3′
Reverse: 5′-CGTTCGCCAGCCTCTTGTCC-3′
Or83b:
Forward: 5′-CCAGAAAGAACAGCTTCCTCATCT-3′
Reverse: 5′-CGAGTCGGATGCTCGTTACC-3′
Rp49:
Forward: 5′-ACTCAATGGATACTGCCCAAGAA-3′
Reverse: 5′-CAAGGTGTCCCACTAATGCATAAT-3′
3
Quantitative Analysis of Target Gene Expression
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Reverse transcription was performed using the RevertAid first-strand cDNA synthesis kit following the manufacturer’s instructions (MBI Fermentas, Vilnius, Lithuania). RNA quantity and quality were assessed by determination of the optical density at 260 and 280 nm using spectrophotometry and additionally by visualization in agarose gels. Real-time PCR was performed in the ABI 7000 SDS (Applied Biosystems) using the Power SYBR Green (Applied Biosystems) according to the manufacturer’s instructions. The thermal cycling conditions comprised of 5 min incubation at 95 °C, followed by 40 cycles at 95 °C for 15 s, 60 °C for 30 s. All of the reactions were performed in triplicate. The relative quantity of the target mRNA was normalized to the level of the internal control GAPDH mRNA level. The relative quantitative analyses of the data were performed using SDS 7000 system software v1.2.3 (Applied Biosystems, USA). The relative quantitation of target gene expression was obtained using the comparative ΔΔCT method [28 (link)].
4
Osteoclast Differentiation Gene Expression
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Real-time quantitative polymerase chain reaction (RT-PCR) was operated using ABI 7000 SDS incorporating Taqman probe mixture (Applied Biosystems). Probes/primes for the genes of interest related to osteoclast differentiation were designed with Primer Express 2.0 software. The oligonucleotide probes were bound with the reporter dye 6-carboxyfluorescein at its 5′ end and the quencher dye tetramethylrhodamine at 3′ terminus. The internal control was the housekeeping gene GAPDH. The PCR reaction was the routine 45 cycles. The expression levels of the altered genes were identified with reference to the changes in comparison to the absolute expression levels of mRNA in the control bone tissues. RT-PCR results were finally computed using the comparative Ct method normalized against the internal housekeeping gene.
Primer sequences were as follows: Bruton Kinase: forward, 5′-GAAGCTGGTGCAGTTGTATG-3′ and reverse, 5′-TATACCCTCTCTGATGCCAG-3′; RANK: forward, 5′-TTAAGCCAGTGCTTCACGGG-3 and reverse, 5′-ACGTAGACCACGATGATGTCGC-3′; NFATc1: forward, 5′- GGAAGGGCGGCTTCTGCGAC-3′ and reverse, 5′- AGGCGTGCGGGCGCAGCAG-3′; NF-κB: forward, 5’-TTTTCGACTACGCAGTGACG-3′ and reverse 5’-GTCCAGAAGGCTCAGGTCAG-3′; Activin A: forward, 5′- CTCGGAGATCATCACGTTTG-3′ and reverse 5-CCTTGGAAATCTCGAAGTGC-3′; GAPDH: forward, 5′-CGACCACTTTGTCAAGCTCA-3′ and reverse, 5′-TTACTCCTTGGAGGCCATGT-3′
Primer sequences were as follows: Bruton Kinase: forward, 5′-GAAGCTGGTGCAGTTGTATG-3′ and reverse, 5′-TATACCCTCTCTGATGCCAG-3′; RANK: forward, 5′-TTAAGCCAGTGCTTCACGGG-3 and reverse, 5′-ACGTAGACCACGATGATGTCGC-3′; NFATc1: forward, 5′- GGAAGGGCGGCTTCTGCGAC-3′ and reverse, 5′- AGGCGTGCGGGCGCAGCAG-3′; NF-κB: forward, 5’-TTTTCGACTACGCAGTGACG-3′ and reverse 5’-GTCCAGAAGGCTCAGGTCAG-3′; Activin A: forward, 5′- CTCGGAGATCATCACGTTTG-3′ and reverse 5-CCTTGGAAATCTCGAAGTGC-3′; GAPDH: forward, 5′-CGACCACTTTGTCAAGCTCA-3′ and reverse, 5′-TTACTCCTTGGAGGCCATGT-3′
5
Temporal Expression of T3S Substrates in C. trachomatis
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The expression of the newly identified candidate T3S substrates during the developmental cycle of C. trachomatis L2/434 was estimated by determining mRNA levels at different times post-infection by real-time quantitative PCR (RT-qPCR). These experiments were done as previously described [45 (link)]. Primers (available upon request) were designed using Primer Express (Applied Biosystems). The RT-qPCR assays were done using the ABI 7000 SDS, SYBR green chemistry, and optical plates (Applied Biosystems), as previously described [52 (link)]. At each time point, raw RT-qPCR data for each gene were normalized against the data obtained for the 16S rRNA transcript, as it was previously demonstrated that this is an adequate endogenous control [52 (link)]. The final results were based on three independent experiments.
6
Quantitative RT-PCR Analysis of Autophagy and Mitochondrial Genes in Drosophila Hearts
7
Real-Time PCR Gene Expression Analysis
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Reverse transcription was performed using the RevertAid first-strand cDNA synthesis kit following the manufacturer’s instructions (MBI Fermentas, Vilnius, Lithuania). RNA quantity and quality were assessed by determination of the optical density at 260 and 280 nm using spectrophotometry and additional visualization by agarose gel electrophoresis. Real-time PCR was performed in the ABI 7000 SDS (Applied Biosystems, Foster City, CA, USA) using the Power SYBR Green (Applied Biosystems) according to the manufacturer’s instructions. Briefly, cDNA was diluted 5 times and 4 μl diluted cDNA template was used for each PCR with 250 nM forward and reverse primers in a total volume of 20 μl. The thermal cycling conditions comprised 5 min at 95°C, followed by 40 cycles at 95°C for 15 s, 60°C for 30 s. All of the reactions were performed in triplicate. The relative quantity of the target mRNA was normalized to the level of the internal control GAPDH mRNA level. The relative quantitative analyses of the data were performed using 7000 system SDS software v1.2.3 (Applied Biosystems). The relative quantitation of target gene expression was obtained using the comparative ΔΔCT method.
8
Splenic RNA Extraction and qPCR Analysis
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Five splenic cryosections (12 μm) per spleen were lysed in QIAzol lysis reagent and total RNA was extracted using the RNeasy® Plus Universal Mini Kit (Qiagen). RNA quantity was determined using the Quantus fluorometer (Promega Biosystems). Translation of 800 ng of total RNA into cDNA was performed using 200 ng of random primer, 0.01 M DTT, 1 μl reaction buffer, 0.5 mM dNTP (each obtained from Promega), and 100 U reverse transcriptase Superscript II RNase H Minus (Invitrogen Life Technologies) in a total volume of 20 μl. Samples were incubated at 42 °C for 50 min. Messenger RNA expression levels were determined by quantitative real-time PCR (qPCR) using the SDS ABI 7000 or SDS ABI 7900 system (Applied Biosystems). Relative abundances of target gene transcripts in a given sample were calculated as differences in cycle of threshold (CT) compared with the geomean expression of the four independent housekeeping genes β-actin, gapdh, mln51 and hprt (ΔCT), and normalized to the ‘sleep’ group (ΔΔCT). Primer and probe sequences as well as gene accession numbers are provided upon request.
9
Quantifying mRNA Expression by qPCR
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Messenger RNA expression levels were measured by quantitative real-time RT-PCR (qPCR) using the SDS ABI 7000 or SDS ABI 7900 system (Applied Biosystems, Foster City, CA, USA). First, 800ng of total RNA were translated into cDNA using 200ng of random primer (Promega), 0.01M DTT, 1μl reaction buffer, 0.5mM dNTP (each obtained from Promega), and 100U reverse transcriptase Superscript II RNase H Minus (Invitrogen Life Technologies) in a total volume of 20μl. Samples were incubated at 42°C for 50min. Primer and probe sequences as well as gene accession numbers are provided on request. Relative abundances of target gene transcripts in a given sample were first calculated as differences in CT compared with the geomean of four independent housekeeping genes, β-ACTIN, GAPDH, MLN51 and HPRT (delta-CT), and then relative to control (delta-delta-CT).
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