Gene expression levels of Bax, Bcl-2, and caspase-3 in heart tissue samples were determined by RT-qPCR on a Roche Light Cycler® 96 System (Roche Life Science, Sandhofer-Germany) and qPCR master mix (QuantiFast SYBR Green PCR Kit). Briefly, total RNA was isolated using RNX-plus reagent (Cinnagen, Iran), according to the manufacturer’s protocol`. The quantity and quality of the isolated RNA were evaluated by NanoDropTM (Thermo Fisher Scientific-USA) and 1% agarose gel electrophoresis, respectively. cDNA was synthesized by reverse transcription of 500 ng of total RNA using PrimeScript first Standard cDNA Synthesis Kit (Takara Biotechnology, Japan). Gene-specific primers (Table 1 ) were designed using AlleleID®6 software (Premier Biosoft Corporation, USA). Online primer blast software (https://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) was used to confirm specificities of designed primers to the corresponding genes. β-actin was considered as housekeeping gene and relative mRNA expression levels (fold change) were calculated by 2 -∆∆Ctformula based on Livak method24 (link) compared with the expression of β-actin.
Quantifast sybr green pcr kit
Manufactured by Roche
Sourced in United States
The QuantiFast SYBR Green PCR kit is a real-time PCR reagent designed for fast and reliable quantification of DNA and RNA targets. The kit includes a fast-cycling SYBR Green PCR master mix that enables rapid cycling protocols.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Lightcycler 96 system, by Roche (2 mentions)
Revertaid first strand dna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Abi 7300 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Rnx plus reagent, by CinnaGen (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Abi prism 7300 pcr machine, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Abi prism 7300, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 system, by Roche (1 mentions)
Fastprep 24, by MP Biomedicals (1 mentions)
8 protocols using quantifast sybr green pcr kit
1
Evaluating Apoptotic Markers in Heart Tissue
2
Ox-LDL-Induced ABCA1 Expression
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Cells, which had been previously treated with compounds 1 , 2 (100 µM) or PE (500 µg/mL) for 2 h, were stimulated by Ox-LDL (50µg/mL) for 24 h. According to the manufacturer’s instructions, total RNA was isolated using TRIzol reagent (Invitrogen Co., Carlsbad, CA, USA). The concentration of RNA was measured by a spectrophotometer, and the cells with an OD value (A260/A280) between 1.8-2.0 were reverse transcribed into cDNA using the synthesis kits (iScript™ cDNA Synthesis Kit; Bio-Rad). Quantitative RT-PCR was performed using a QuantiFast SYBR® Green PCR Kit (Roche) and ABI PRISM 7300 PCR Machine (Applied Biosystems). The cDNA was amplified at 90°C for 10 min, 40 cycles at 95°C for 15 s, 60°C for 60 s. Final extension was performed for 7 min at 72°C. The PCR products were electrophoresed on 2% agarose gel and stained with ethidium bromide. PCR primers were as follows: ABCA1, 5’-GCAGATCA AGCATCCCAAC T-3’ (forward) and 5’-CCAGAGAATGT TTCATTGTCCA-3’ (reverse).
3
RNA Extraction and qRT-PCR Analysis
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The RNA extraction was performed utilizing TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in compliance with manufacturer’s protocols. The RevertAid™ First Strand DNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for reverse transcription. qRT-PCR was conducted with QuantiFast SYBR-Green PCR kit (Roche, Indianapolis, IN, USA) on ABI 7300 Real-time PCR System (Applied Biosystems, Foster City, CA, USA) with cDNA working as the template. The gene relative expressions were determined utilizing 2-ΔΔCT analysis, and the primers were synthesized by BGI (Shenzhen, China) with GAPDH and U6 working as the endogenous controls.
4
Quantitative Expression Analysis of GATA6-AS1, miR-19a-5p, and TET2
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Total RNA was extracted from cell lines or tissues using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and was reverse transcribed into cDNA using a RevertAid™ First Strand DNA Synthesis kit (Thermo Fisher Scientific, Inc.). QuantiFast SYBR-Green PCR kit (Roche Diagnostics) was used to perform qPCR on an ABI 7300 Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling protocol was as follows: Initial denaturation at 95°C for 5 min, followed by 45 repeats of a three-step cycling program consisting of 10 sec at 95°C (denaturation), 10 sec at 60°C (primer annealing) and 10 sec at 72°C (elongation), and a final extension step for 10 min at 72°C. The sequences of the primers were as follows: GATA6-AS1, forward 5′-ACCACAACCACTACCTTATGGCGT-3′, reverse 5′-TGCCATCTGGACTGCTGGACAATA-3′; miR-19a-5p, forward 5′-GTTTGCTGGGAAGGCAAAG-3′, reverse 5′-TGTTTTGCTGGGAAGGCAAA-3′; U6, forward 5′-CGCTTCGGCAGCACATATAC-3′, reverse 5′-TTCACGAATTTGCGTGTCAT-3′; TET2, forward 5′-GGACTGAGCTGCTGAATTCAACT-3′, reverse 5′-CCTCAACATGGTTGGTTCTATCC-3′; and GAPDH, forward 5′-TGTCCGTCGTGGATCTGA-3′ and reverse 5′-TTGCTGTTGAAGTCGCAGGAG-3′. The relative expression levels of GATA6-AS1, miR-19a-5p and TET2 were normalized to endogenous controls GAPDH or U6 and were expressed as 2−ΔΔCq (20 (link)).
5
Quantitative RT-PCR Analysis of Gene Expression
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Total RNA from RAW264.7 macrophages and heart tissue was isolated using TRIzol reagent (Takara Bio. Inc., Kyoto, Japan) according to the manufacturer’s recommendations. RNA purity was assessed by the 260/280 nm ratio, and total RNA (2 μg) was reverse-transcribed into cDNA using a reverse transcription assay (Promega, Madison, WI, USA) in 25-μL reaction volumes according to the manufacturer’s instructions. Real-time polymerase chain reaction (PCR) was performed with a QuantiFast SYBR® Green PCR Kit (Roche Diagnostics, Indianapolis, IN, USA) on an ABI Prism 7300 sequence detection system (Applied Biosystems, Foster City, CA, USA). The primer sequences used for real-time PCR are shown in Table 1 . The conditions for amplification were as follows: 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 60 s. Relative expression of real-time PCR products was normalized to the expression of β-actin and expressed as the transcript fold change relative to that of the control group using the 2−ΔΔCt method [22 (link)].
6
Quantification of Impure DNA in MC Products
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The presence of impure DNAs (PBs and PPs) in the MC products was determined by absolute quantitative qPCR. The MC producer plasmid PP5 was used as a standard. The PCR primers kan-F (5′- GCCCAATAGCAGCCAGTCC) and kan-R (5′-TGATGCCGCCGTGTTCC) (Supplementary Table S1 ) were specific for kanamycin resistance gene shared by all PPs. qPCR was performed using the Qiagen QuantiFast SYBR Green PCR kit and the machine of Roche system 480 (Shanghai, China). The PCR program included a denaturing step of 94 °C for 5 minutes, followed by 40 cycles of 94 °C for 20 seconds, and 68 °C for 20 seconds each. Under these experimental conditions, the correlation equation was CT = −3.1226 lgX0 + 45.26 (R2 = 0.9988), where X0 is the copy number of DNA.
7
Quantitative ChIP-qPCR for Gene Expression
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ChIP-qPCR was performed using QuantiFast SYBR Green PCR kit according to the manufacturer’s instructions and each reaction was performed in triplicates using a Roche LightCycler 480 system. The results were analyzed with absolute quantification/2nd derivative maximum and the 2(-ΔC(t)) method as previously described (Livak and Schmittgen, 2001 (link)). Error bars represent standard deviations.
8
ChIP-qPCR Protocol for Protein-DNA Interactions
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Chromatin immunoprecipitation (ChIP) was carried out as previously described (6) .
Briefly, cells were collected at the indicated experimental conditions and crosslinked with 1% formaldehyde for 30 min. Cells were washed twice with ice-cold 1X TBS, suspended in lysis buffer supplemented with 1 mM PMSF, 20 mM NEM, and 1X
EDTA-free complete cocktail, and lysed using FastPrep-24 (MP Biomedicals).
Chromatin was sheared to a size of 300-500 bp by sonication. IP reactions with anti-HA antibodies and Dynabeads protein G were allowed to proceed overnight at 4°C.
After washing and eluting the ChIP fractions from beads, crosslinks were reversed at 65°C overnight for both Input and IP. After proteinase K treatment, DNA was extracted twice by phenol/chlorophorm/isoamyl alcohol (25:24:1, v/v). Following precipitation with ethanol and Ribonuclease A (RNase A) treatment, DNA was purified using QIAquick PCR purification kit. Real-time PCR was performed using QuantiFast SYBR Green PCR kit according to the manufacturer's instructions and each reaction was performed in triplicates using a Roche LightCycler 96 system. The results were analyzed with absolute quantification/2 nd derivative maximum and the 2(-ΔC(t)) method. Each ChIP experiment was repeated at least three times. Statistical analysis was performed using Student's unpaired t-test. The error bars represent standard error of mean (SEM).
Briefly, cells were collected at the indicated experimental conditions and crosslinked with 1% formaldehyde for 30 min. Cells were washed twice with ice-cold 1X TBS, suspended in lysis buffer supplemented with 1 mM PMSF, 20 mM NEM, and 1X
EDTA-free complete cocktail, and lysed using FastPrep-24 (MP Biomedicals).
Chromatin was sheared to a size of 300-500 bp by sonication. IP reactions with anti-HA antibodies and Dynabeads protein G were allowed to proceed overnight at 4°C.
After washing and eluting the ChIP fractions from beads, crosslinks were reversed at 65°C overnight for both Input and IP. After proteinase K treatment, DNA was extracted twice by phenol/chlorophorm/isoamyl alcohol (25:24:1, v/v). Following precipitation with ethanol and Ribonuclease A (RNase A) treatment, DNA was purified using QIAquick PCR purification kit. Real-time PCR was performed using QuantiFast SYBR Green PCR kit according to the manufacturer's instructions and each reaction was performed in triplicates using a Roche LightCycler 96 system. The results were analyzed with absolute quantification/2 nd derivative maximum and the 2(-ΔC(t)) method. Each ChIP experiment was repeated at least three times. Statistical analysis was performed using Student's unpaired t-test. The error bars represent standard error of mean (SEM).
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