Total RNA was extracted from tissues and cell lines using the TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed at 65°C for 120 sec using the M-MLV RT kit (Promega Corporation, Madison, WI, USA) following the manufacturer's protocol. The expression of miR-338-3p was quantified using TaqMan microRNA assays (Ambion; Thermo Fisher Scientific, Inc.) with the ABI 7900 Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The mRNA expression of MACC1 was detected using SYBR® Green qPCR SuperMix (Invitrogen; Thermo Fisher Scientific, Inc.) and quantified using ABI 7900 Sequence Detection System. Amplification was performed using the following thermocycling protocol: Preheating at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 5 sec and annealing/extension at 60°C for 20 sec. The primers used for the current study were as follows: miR-338-3p forward 5′-TGCGGTCCAGCATCAGTGAT-3′ and reverse 5′-CCAGTGCAGGGTCCGAGGT-3′; U6 forward 5′-GCTCGCTTCGGCAGCACA-3′ and 5′-GAGGTATTCGCACCAGAGGA-3′; MACC1 forward 5′-CCTTCGTGGTAATAATGCTTCC-3′ and reverse 5′-AGGGCTTCCATTGTATTGAGGT-3′; GAPDH forward 5′-ACCACAGTCCATGCCATCCAC-3′ and reverse 5′-TCCACCACCCTGTTGCTGTA-3′. The relative expression of miR-338-3p or MACC1 was normalized to U6 small nuclear RNA or GAPDH, respectively and calculated using the 2−ΔΔCq method (22 ).
Abi 7900 sequence detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
The ABI 7900 Sequence Detection System is a real-time PCR instrument designed for quantitative gene expression analysis. It features a 96-well block format and supports fluorescent detection of multiple targets in a single reaction. The system provides precise and reliable data for a wide range of applications, including gene expression profiling, SNP genotyping, and viral load quantification.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (28 mentions)
Primescript rt reagent kit, by Takara Bio (8 mentions)
Sybr green 1 dye, by Thermo Fisher Scientific (7 mentions)
Rneasy kit, by Qiagen (6 mentions)
Rt pcr kit, by Thermo Fisher Scientific (4 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Sybr premix ex taq, by Takara Bio (4 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (4 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Sybr primescript ready mix, by Takara Bio (3 mentions)
Goscript reverse transcription system, by Promega (3 mentions)
Sybr green master mix, by Thermo Fisher Scientific (3 mentions)
Sybr green master mix kit, by Takara Bio (3 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
Taqman microrna assay, by Thermo Fisher Scientific (3 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Primer express 2, by Thermo Fisher Scientific (3 mentions)
Superscript 3 cdna synthesis system, by Thermo Fisher Scientific (2 mentions)
95 protocols using abi 7900 sequence detection system
1
Quantifying miR-338-3p and MACC1 Expression
2
Quantification of miR-506 and ZEB2 Expression
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Total RNA from cell lines and tissues was extracted with TRIzol® reagent (Invitrogen), according to the manufacturer’s instructions. The concentration of the total RNA was quantified using a NanoDrop-2000 (Peqlab). cDNA was synthesized with the TaqMan MicroRNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) and expression was determined using the QuantMir RT Kit (System Biosciences, Mountain View, CA, USA) and ABI 7900 Sequence Detection System (Applied Biosystems) using miR-506 and U6 primers (Applied Biosystems). To evaluate ZEB2 mRNA expression, RNA was reverse-transcribed into cDNA by the Primescript™ RT reagent kit (Takara, Dalian, China) and examined with the Real-time PCR Mixture Reagent (Takara) and ABI 7900 Sequence Detection System (Applied Biosystems), using primers for ZEB2 and α-tubulin. The relative miR-506 expression was normalized to U6 expression and the expression of ZEB2 mRNA was normalized to α-tubulin using the 2-ΔΔCt method. Values of genes were first normalized against U6 or α-tubulin, and then compared to the experimental controls.
3
Quantitative RT-PCR for Gene Expression
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Total RNA was extracted immediately after retrieval from renal cortical tissues using the TRIzol Reagent according to the manufacturer's instructions. Two micrograms of total RNA was reverse‐transcribed using the superscript III first‐strand synthesis system (Invitrogen, Carlsbad, CA). Real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) was performed using a SYBR green dye I (Applied Biosystems, Foster City, CA) with the ABI 7900 Sequence Detection System (Applied Biosystems). cDNA was first denatured at 95°C for 15 min and then amplified through 40 amplification cycles each consisted of denaturation at 95°C for 15 sec and annealing/extension at 60°C for 60 sec, according to the manufacturer's protocol. Fluorescence signals were recorded in each cycle. Relative quantification of gene expression was carried out using the 2ΔΔCT method and analyzed with RQ‐manager 1.2. (ABI 7900 Sequence Detection System; Applied Biosystems). Samples were run in triplicate in separate tubes to permit quantification of the target gene normalized to β‐actin. Sequences of primers used are listed in Table 1 . The specificity of the PCR products was confirmed on a 1.5% agarose gel by showing a specific single band with the expected size.
4
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA was extracted from WT and Bloc1s2−/− dissected cortices at E14.5 with RNeasy (Qiagen) and used to generate first-strand cDNA with the Superscript III cDNA synthesis system (Invitrogen) according to the manufacturer’s instructions. qRT-PCR analysis was performed using SYBR PrimeScript Ready Mix (Takara) in an ABI 7900 sequence detection system (Applied Biosystems). GAPDH expression was used for normalization. Total RNAs are from the dissected trunk region of zebrafish embryos. The PCR primers are listed in Supplementary file 2 .
5
Real-Time RT-PCR Analysis of Renal Fibrosis
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Total RNA was extracted from renal cortex tissue using Tri Reagent according to the manufacturer’s instructions. Two micrograms of total RNA was reverse-transcribed using the superscript III first-strand synthesis system for RT-PCR kit (Invitrogen). Real-time RT-PCR was performed using the SYBR green dye I (Applied Biosystems, Foster City, CA, U.S.A.) and the ABI 7900 Sequence Detection System (Applied Biosystems) as described previously [34 (link)]. Samples were run as triplicates in separate tubes to permit quantification of the target gene normalized to β-actin. Sequences of primers used for fibrotic markers such as TGFβ1, PAI-1, FN, Col-I, Col-III, Col-IV, and GAPDH were described previously [31 (link),35 (link)].
6
Genotyping of Two Selected SNPs
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Peripheral blood of 2 ml was collected from each participant. Genomic DNA was extracted from human peripheral blood cells using the FlexiGene DNA Kit 250 (Qiagen, Hilden, Germany) according to the manufactures’ guidelines. Two selected SNPs (rs6832151 and rs9355610) were genotyped using TaqMan assays on ABI7900 platform. TaqMan SNPs genotype assays were provided by Applied Biosystems (C__29224385_10 for rs6832151 and C__30614352_10 for rs9355610, respectively). SNP genotyping was performed on ABI7900 Sequence Detection System (Applied Biosystems, USA) according to the manufacturer’s instructions. The data completion rate of rs6832151 and rs9355610 was 99.6% and 99.4% respectively.
7
Quantitative Methylation-Specific PCR for CAV1
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DNA was treated with bisulfite to convert unmethylated cytosines to uracils prior to qMSP, as described previously
[27 (link)]. Promoter methylation levels of CAV1 were determined with the ABI 7900 Sequence Detection System (Applied Biosystems, Foster City, CA), using primers and probes as described previously
[27 (link)]. A standard curve was generated using serial dilutions of CpGenome Universal Methylated DNA (CHEMICON, Temecula, CA). The normalized methylation value (NMV) was defined as follows: NMV = (CAV1-S/CAV1-FM)/(ACTB-S/ACTB-FM), where CAV1-S and CAV1-FM represent the methylation levels of CAV1 in sample and universal methylated DNAs, respectively, while ACTB-S and ACTB-FM correspond to ß-Actin in sample and universal methylated DNAs, respectively
[21 (link)].
[27 (link)]. Promoter methylation levels of CAV1 were determined with the ABI 7900 Sequence Detection System (Applied Biosystems, Foster City, CA), using primers and probes as described previously
[27 (link)]. A standard curve was generated using serial dilutions of CpGenome Universal Methylated DNA (CHEMICON, Temecula, CA). The normalized methylation value (NMV) was defined as follows: NMV = (CAV1-S/CAV1-FM)/(ACTB-S/ACTB-FM), where CAV1-S and CAV1-FM represent the methylation levels of CAV1 in sample and universal methylated DNAs, respectively, while ACTB-S and ACTB-FM correspond to ß-Actin in sample and universal methylated DNAs, respectively
[21 (link)].
8
Quantitative gene expression analysis
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Total RNA was isolated using Trizol reagent and then reverse transcribed to complementary DNA (cDNA) using the PrimeScript RT Reagent Kit (Takara, Dalian, China). Real-time PCR was carried out on an ABI 7900 sequence detection system (Applied Biosystems, Foster City, CA, USA) using SYBR Premix Ex Taq (Takara). GAPDH was used as the reference gene. Relative expression levels were calculated using the 2−ΔΔCT method, and all experiments were conducted in triplicate.
9
ChIP Assay for Telomere and Subtelomeric Region Analysis
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ChIP assays were performed with the protocol provided by Millipore with minor modifications as described previously (Deng et al. 2009 (link)). Briefly, LCLs were crosslinked in 1% formaldehyde with shaking for 15 min, and DNA was sheared to between 200- and 400-bp fragments by sonication with a Diagenode Bioruptor. Quantification of ChIP DNA at subtelomeric regions was determined using quantitative PCR (qPCR) with the ABI 7900 sequence detection system (Applied Biosystems). qPCR was performed in triplicates from three independent ChIP experiments, and PCR data were normalized to input values. Primer sequences used for qPCR were designed using Primer Express (Applied Biosystems), and listed in Supplemental Table 10. Each primer sets was validated by using melting curve analysis, in which one major dissociation peak was observed. ChIP DNA at telomeres was assayed by dot blotting with γ-[32P]ATP-labeled probes specific for telomere (4 × TTAGGG) or Alu repeats (cggagtctcgctctgtcgcccaggctggagtgcagtggcgcga). After hybridization, the blot was developed with a Typhoon 9410 imager (GE Healthcare) and quantified with ImageQuant 5.2 software (Molecular Dynamics). Antibodies used in ChIP assay include rabbit polyclonal antibodies to CTCF (Millipore 07-729) and RAD21 (abcam ab992). Rabbit antibodies to TERF1 and TERF2 were generated against recombinant protein and affinity purified.
10
Quantitative Analysis of PELI2 mRNA Expression
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Total RNA from cells was extracted using TRIzol (Invitrogen; Thermo Fisher Scientifc, Inc.). For the reverse transcription, cDNA were generated by a Primescript™ RT reagent Kit (Takara, Dalian, China). qRT-PCR was carried out using SYBR Green PCR Master Mix (Takara) and monitored using an ABI 7900 sequence detection system (Applied Biosystems, CA, U.S.A.). The amplification steps were as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 30 s, 60°C for 40 s, and 72°C for 1 min. β-Actin was used as an internal control. The mRNA expression was calculated by the 2−ΔΔCT method. The following primer sequences were used: PELI2: forward, 5′-CGC GCG CGG ATT TGA CTC TT-3′, reverse, 5′-CTG GGT GAA GCC CCC TCG TG-3′; β-actin: forward, 5′-TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-3′, reverse, 5′-CTA GAA GCA TTT GCG GTG GAC GAT GGA GGG-3′.
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