The relative expression of TUBG1 mRNA and lncRNA-MALAT1 in liver tissue were measured using a Quantitect SYBR Green Master Mix kit and an RT2 SYBR Green ROX real time-polymerase chain reaction (qPCR) Mastermix (Qiagen), respectively, using a 7500 qPCR Systems (Applied Biosystems, Foster City, CA, United States) detection system) and specific primers (Accession: NM_001128148, NM_003234, NR_002819, and ENST00000534336, respectively) supplied by Qiagen. GAPDH (Accession NM_002046.7) was used as a housekeeping gene. MiR-3163 expression in liver tissue was quantified by PCR using a miScript SYBR Green kit (Qiagen), a miScript universal primer, and a miRNA-specific forward primer (mir-125b-1-3p miScript Primer Assay) (Accession: MIMAT0004592 (5'-ACGGGUUAGGCUCUUGGGAGCU). All steps followed the manufacturer’s suggested protocol, and SNORD68 was used as an internal control. Ct values more than 36 were considered as negative. The specificities of the amplicons for the SYBR Green−based PCR amplification were affirmed by the melting curves. The 2−ΔΔCt technique was used to measure the relative expression of the HCC-specific RNA panel.
Quantitect sybr green master mix kit
Manufactured by Qiagen
Sourced in United States, Germany
The Quantitect SYBR Green master mix kit is a ready-to-use solution for real-time quantitative PCR (RT-qPCR) analysis. The kit contains a proprietary DNA polymerase, SYBR Green I dye, and optimized buffer components to enable sensitive and specific detection of target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Omniscript kit, by Qiagen (4 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (4 mentions)
Primer express, by Thermo Fisher Scientific (3 mentions)
Rnaeasy mini kit, by Qiagen (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Miscript sybr green kit, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Dulbecco s modified eagle s medium dmem, by Bioidea (1 mentions)
Fetal bovine serum (fbs), by Biosera (1 mentions)
Dna engine opticon, by Bio-Rad (1 mentions)
Iscript reverse transcriptase, by Bio-Rad (1 mentions)
Rneasy, by Qiagen (1 mentions)
Mirneasy extraction kit, by Qiagen (1 mentions)
7500 fast system, by Thermo Fisher Scientific (1 mentions)
Miscript sybr green pcr kit, by Qiagen (1 mentions)
9 protocols using quantitect sybr green master mix kit
1
Quantifying Liver Tissue RNA Profiles
2
Quantitative Real-Time PCR for Cytokine mRNA
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One lung lobe, from three different mice per group in two independent experiments, was used to isolate mRNA using the RNAeasy minikit (Qiagen). Quality and quantity of RNA were evaluated through spectrophotometry (260/280) on agarose gels. Reverse transcription of the mRNA was performed using 5 μg RNA, oligo (dT), and the Omniscript kit (Qiagen, Inc.). Real-time PCR was carried out using the 7500 real time PCR system (Applied Biosystems) and Quantitect SYBR green Mastermix kit (Qiagen). Standard curves of quantified and diluted PCR product, as well as negative controls, were included in each PCR run. Specific primers for genes encoding acidic ribosomal protein (RLP0) as house-keeping gene, tumor necrosis factor alpha (TNFα), gamma interferon (IFNγ), interleukin 17 (IL-17) and interleukin 10 (IL-10) were designed using the program Primer Express (Applied Biosystems) [21 (link)]. Cycling conditions were: initial denaturation at 95°C for 15 min, followed by 40 cycles at 95°C for 20 s, 60°C for 20 s, and 72°C for 34 s. Data are shown as copies of cytokine-specific mRNA/106 copies of RLP0 mRNA.
3
Cytokine mRNA Expression Analysis
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Left or right lung lobes from three different mice per group in two different experiments were used to isolate mRNA using the RNeasy Mini Kit (Qiagen), according to recommendations of the manufacturer. Quality and quantity of RNA were evaluated through spectrophotometry (260/280) and on agarose gels. Reverse transcription of the mRNA was performed using 5 μg RNA, oligo-dT, and the Omniscript kit (Qiagen, Inc). Real-time PCR was performed using the 7500 real time PCR system (Applied Biosystems, USA) and Quantitect SYBR Green Mastermix kit (Qiagen). Standard curves of quantified and diluted PCR product, as well as negative controls, were included in each PCR run. Specific primers for genes encoding acidic ribosomal protein (RLP0) as house keeping gene (FWD: 5′-CTC TCG CTT TCT GGA GGG TG-3′; RV: 5′-ACG CGC TTG TAC CCA TTG AT-3′), TNF-α, IFN-γ, IL-12, iNOS, IL17, were designed using the program Primer Express (Applied Biosystems, USA) [24] .
Cycling conditions used were: initial denaturation at 95°C for 15 min, followed by 40 cycles at 95°C for 20 sec, 60°C for 20 sec, 72°C for 34 sec. Quantities of the specific mRNA in the sample were measured according to the corresponding gene specific standard. The mRNA copy number of each cytokine was related to one million copies of mRNA encoding the RLP0 gene [25] (link).
Cycling conditions used were: initial denaturation at 95°C for 15 min, followed by 40 cycles at 95°C for 20 sec, 60°C for 20 sec, 72°C for 34 sec. Quantities of the specific mRNA in the sample were measured according to the corresponding gene specific standard. The mRNA copy number of each cytokine was related to one million copies of mRNA encoding the RLP0 gene [25] (link).
4
Quantitative RT-PCR Analysis of FFPE Samples
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According to the manufacturer’s instructions, total mRNA from paraffin-embedded tissues was obtained with The NucleoSpin totalRNA FFPE kit for RNA extraction (Macherey-Nagel Thermo Fisher Scientific, Waltham, MA, USA). The quality and quantity of RNA were evaluated through spectrophotometry (260/280) and on agarose gels. Reverse mRNA transcription was performed using 2 ng RNA, oligo (dT), and the Omniscript kit (Qiagen, Hilden, Germany). RT-PCR was carried out using the 7500 real-time PCR system (Applied Biosystems Inc., Foster City, CA, USA) and Quantitect SYBR Green Master Mix kit (Qiagen, Hilden, Germany). Specific primers were designed using the program Primer 359 Express (Applied Biosystems, Waltham, MA, USA), for the following targets: RPLP0, housekeeping gene (ribosomal protein lateral stalk subunit P0), TNFα F: 5-CTCTCGCTTTCTGGAGGGTG-3; R: 5-357 ACGCGCTTGTACCCATTGAT-3, IFN-γ F: 5-GGTGACATGAAAATCCTGCAG-3; R: 5-CCTCAAACTTGGCAATACTCATGA-3. Initial denaturation at 95 °C for 15 min was followed by 40 cycles at 95 °C for 20 s, 60 °C for 20 s, and 72 °C for 34 s. Each sample was examined twice. The 2−(∆∆Ct) technique calculates the fold change in gene expression [52 (link)].
5
Quantitative Analysis of Immune Transcripts
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Three lungs from each of the groups were used to isolate mRNA using the RNAeasy minikit (Qiagen, Valencia, CA, USA). The quality and quantity of RNA were evaluated through spectrophotometry (260/280) and on agarose gels. Reverse transcription of the mRNA was performed using 100 ng RNA, oligo (dT), and the Omniscript kit (Qiagen, Valencia, CA, USA). Real-time polymerase chain reaction (PCR) was carried out using the 7500 real-time PCR system (Applied Biosystems) and Quantitect SYBR green Mastermix kit (Qiagen, Valencia, CA, USA). Standard curves of quantified and diluted PCR products, as well as negative controls, were included in each PCR run. Specific primers for genes encoding the ribosomal protein, large P0 (RPLP0) as house-keeping gene, IL-4, IFN-ϒ, TGF-β, TNF-α, IDO, HO-1, IL-22, or IL-17 were designed using the program Primer Express (Applied Biosystem, San Francisco, CA, USA) (Table 1
6
Cytotoxicity and Antioxidant Evaluation
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PCA was obtained from Cayman Chemical Co. (USA; Cat No. 14916-25) as a synthetic material with ≥ 98% purity. DOX was prepared from EBEWE Pharma GmbH Nfg KG Co. (Austria; Cat No. 118529), and ATO from Bahar Sabz Talaie Pharmacy (Iran; Global Trade Item No. 06262749061436). Fetal bovine serum (FBS) was purchased from Biosera Co. (France). Dulbecco's modified eagle's medium (DMEM) was obtained from BioIdea Co. (Iran). MTT (3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay kit was purchased from Alfa Aesar GmbH & Co KG (Germany). The assay kits for the determination of total oxidant capacity and ferric-reducing antioxidant power (FRAP) were prepared by Hakiman Shargh Research Co. (Iran). The lactate dehydrogenase (LDH) release assay kit was from Kiazist (Iran). BIOFACTTM total RNA Prep kit was purchased from BioFact Ltd. (Korea), the cDNA synthesis kit from Yekta Tajhiz Azma Co. (Iran), and the Quantitect SYBR Green master mix kit from Qiagen (Germany). All other chemicals were from Merck Co., Germany.
7
Quantitative PCR Analysis of Gene Expression
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RNA was extracted from 0.5 × 105 to 1 × 107 cells using the RNeasy protocol, as recommended by the manufacturer (RNeasy, Qiagen, Valencia CA, USA), and cDNA produced using a mixture of random hexamers and oligo dT priming (iScript reverse transcriptase, Biorad, Hercules, CA, USA). Quantitative PCR was carried out in triplicate using the Quantitect SYBR Green master mix kit (Qiagen, Valencia, CA, USA) according to the manufacturers recommendations, in an Opticon DNA Engine (MJ Research, Reno, NV, USA). Primer sequences used are as follows:
c-myc F: 5′-TCGGATTCTCTGCTCTCCTCG-3′
c-myc R: 5′-CTGCGTAGTTGTGCTGATGTGTG-3′
p21 F: 5′-ACAGCAGAGGAAGACCATGTGG-3′
Line-1 F: 5′-GCTGGATATGAAATTCTGGGTTGA-3′
Line-1 R: 5′-AGGAAATACAGAGAACGCCACAA-3′
CrabpI F: 5′-GGACGCAAGTGCAGGAGTTTA-3′
CrabpI R: 5′-GCGCCAAACGTCAGGATAA-3′
GAPDH F: 5′-CCAAAATCAAGTGGGGCGATG-3′
GAPDH R: 5′-AAAGGTGGAGGAGTGGGTGTCG-3′.
8
Pancreatic RNA Extraction and Profiling
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Total RNA, involving mRNAs and miRNAs, extraction from the 60 mg of frozen pancreas tissue samples was performed using a miRNEasy extraction kit (Qiagen, Hilden, Germany, Cat. No. 217004) according to the protocol supplied with the kit. NanoDrop (Thermo scientific, USA) was utilized to assess the concentration and purity of total RNA and the purity of the isolated RNAs was adjusted to be 1.8–2 (A260/A280). The RNA extracted from the pancreas tissues was then reverse transcribed into complementary DNA using miScript II RT (Cat. No. 218161, Qiagen, Germany).
Relative expression of the selected RNAs species in the pancreatic tissue samples was assessed using a Quantitect SYBR Green Master Mix Kit (Qiagen, Germany, Cat. No. 204143) for DDX58, NFκB1, and CHUK mRNAs and miScript SYBR Green PCR Kit (Qiagen, Germany, Cat no. 218073) for miR-1976 miRNA. Real-time (RT)-qPCR was conducted on 7500 Fast System (Applied Biosystems, Foster City, USA). The GAPDH and SNORD72 were used as housekeeping genes. The primers list used herein was obtained from Qiagen, Germany (Additional file1 : Table S1). The relative quantification of RNA expression was calculated using RQ = 2 –ΔΔCt formula [64 (link)].
Relative expression of the selected RNAs species in the pancreatic tissue samples was assessed using a Quantitect SYBR Green Master Mix Kit (Qiagen, Germany, Cat. No. 204143) for DDX58, NFκB1, and CHUK mRNAs and miScript SYBR Green PCR Kit (Qiagen, Germany, Cat no. 218073) for miR-1976 miRNA. Real-time (RT)-qPCR was conducted on 7500 Fast System (Applied Biosystems, Foster City, USA). The GAPDH and SNORD72 were used as housekeeping genes. The primers list used herein was obtained from Qiagen, Germany (Additional file
9
Quantitative Analysis of TLR4 Expression
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After relevant treatment, extraction of total RNA was performed from H9C2 cells using BIOFACT™ Total RNA Prep kit (BioFact Ltd., Korea). A NanoDrop system spectrophotometer (260/280 nm) was used for approving the concentrations of RNA and their purity. For detecting the expression of TLR4 gene, cDNA was synthesized using a commercial kit (Yekta Tajhiz Azma Co., Iran). Then, Quantitect SYBR Green master mix kit (Qiagen, Germany) on a StepOne™ RT-PCR System (USA) was used for quantitative RT-PCR (real time-polymerase chain reaction) analyzing [23] (link).
Primers were synthesized by Sina Clon Co. (Iran) and their sequences were as follows: TLR4 forward 5'-CACATAGCAGATGTTCCTAG-3' and TLR4 reverse 5'-CCAAAGCTGATATCCTCTC-3'. GAPDH (endogenous glyceraldehyde 3-phosphate dehydrogenase) was considered as an internal control. The relative gene expression was estimated via 2 -ΔΔ Ct procedure.
Primers were synthesized by Sina Clon Co. (Iran) and their sequences were as follows: TLR4 forward 5'-CACATAGCAGATGTTCCTAG-3' and TLR4 reverse 5'-CCAAAGCTGATATCCTCTC-3'. GAPDH (endogenous glyceraldehyde 3-phosphate dehydrogenase) was considered as an internal control. The relative gene expression was estimated via 2 -ΔΔ Ct procedure.
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