The total RNA was extracted using a RNeasy mini kit (Qiagen, 74104) and RNAse-Free DNase set kit (Qiagen, 79254); it was then reverse transcribed into cDNA using a high-capacity cDNA reverse transcription kit (Applied BiosystemsTM, 4368814). The DNA and cDNA quality and quantity were assessed using a NanoPhotometer® (Implen, NP80). Quantitative real-time polymerase chain reactions (PCRs) were performed using a StepOneTM real-time PCR system (Applied BiosystemsTM) and PowerUPTM SYBRTM green master mix (Applied BiosystemsTM, A25918). Glyceraldehyde 3-phosphate dehydrogenase was used as internal control, and the relative gene expression was calculated using the 2−ΔΔCT method. Primers were designed using the GenScript® real-time PCR primer design software or NCBI-BLAST software; the sequences are listed in Supplementary Table 7 .
Rnase free dnase set kit
Manufactured by Qiagen
Sourced in United Kingdom
The RNase-Free DNase Set kit is a laboratory product designed to remove DNA from RNA samples. It contains reagents and buffers necessary for the efficient digestion of DNA. The kit is intended to be used in RNA purification workflows to ensure the removal of any contaminating DNA, enabling the subsequent analysis of pure RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (9 mentions)
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Rneasy kit, by Qiagen (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Qiashredder kit, by Qiagen (2 mentions)
Experion rna stdsens analysis kit, by Bio-Rad (2 mentions)
Rnalater, by Thermo Fisher Scientific (2 mentions)
Poweruptm sybrtm green master mix, by Thermo Fisher Scientific (1 mentions)
Steponetm real time pcr system, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Nanophotometer, by Implen (1 mentions)
Rt2 profiler pcr arrays kit, by Qiagen (1 mentions)
Rneasy minelute cleanup kit, by Qiagen (1 mentions)
Rt2 first strand kit, by Qiagen (1 mentions)
Rt2 sybr green mastermix, by Qiagen (1 mentions)
Rneasy powerplant kit, by Qiagen (1 mentions)
20 protocols using rnase free dnase set kit
1
Quantitative Real-Time PCR Analysis
2
Validation of Differentially Expressed Genes
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Differentially expressed genes identified in the cDNA microarray assay were validated by quantitative RT-PCR assay by using a customized detection system, SyBr Green platform PCR Array® (Qiagen), with 88 target genes, 5 housekeeping genes, and 3 internal control genes. Validation was performed for 24 samples, three of each group (3TT, 3BT, 3BB, 3BL, 3LL, 3R1, 3R2, and 3 controls) including both samples used in the microarray assay and new samples.
The RNA samples were treated with DNase (RNase-Free DNase Set kit, Qiagen) to eliminate residual genomic DNA. Subsequently, RNA was purified and concentrated with the RNeasy MinElute Cleanup Kit (Qiagen) according the manufacturer's specification. The RT2 First Strand Kit (Qiagen) was used for cDNA synthesis. The efficiency of the reverse transcription reaction was assessed by using a RT-PCR reaction for GAPDH.
The ABI ViiA 7 instrument was used for real-time PCR reactions (Applied Biosystems) following the RT2 Profiler PCR Arrays Kit (Qiagen) protocol in combination with RT2 SYBR Green Master mix (Qiagen).
The mRNAs were selected after expression analysis in all comparisons. The authors chose the most up and down regulated present in all groups, and therefore, those who were related to the disease. Others were selected because they were differentially expressed in particular forms of the disease or reaction.
The RNA samples were treated with DNase (RNase-Free DNase Set kit, Qiagen) to eliminate residual genomic DNA. Subsequently, RNA was purified and concentrated with the RNeasy MinElute Cleanup Kit (Qiagen) according the manufacturer's specification. The RT2 First Strand Kit (Qiagen) was used for cDNA synthesis. The efficiency of the reverse transcription reaction was assessed by using a RT-PCR reaction for GAPDH.
The ABI ViiA 7 instrument was used for real-time PCR reactions (Applied Biosystems) following the RT2 Profiler PCR Arrays Kit (Qiagen) protocol in combination with RT2 SYBR Green Master mix (Qiagen).
The mRNAs were selected after expression analysis in all comparisons. The authors chose the most up and down regulated present in all groups, and therefore, those who were related to the disease. Others were selected because they were differentially expressed in particular forms of the disease or reaction.
3
Quantifying mRNA Expression by ddPCR
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The extraction of mRNA was conducted using Trizol reagent (Thermo Fisher 15596026) and RNeasy kit from Qiagen (74104) and RNase-Free DNase Set kit from Qiagen (79254). The cDNA was produced using iScript cDNA Synthesis Kit (BioRad, 1708890). The levels of HIS3 mRNA were measured by ddPCR using HIS3 specific primer sets (see Supplementary Table 4 ) and normalized to ACT1 using the same primer sets as in AMBER.
4
Extraction and Quantification of RNA
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After 1 week, when a significant increase in co-incubated N2 fixation rates was verified (approximately 3 times higher), the cotton stoppers were replaced with the original plastic caps, and the samples were flash-frozen by immersion into liquid N2. The tubes were kept at − 80 °C until they were freeze-dried at − 55 °C for 24 h. The dried samples were moved to RNeasy PowerBead Tubes (Qiagen, Venlo, Netherlands), which had their ceramic beads replaced with 150–212-μm acid-washed glass beads (Sigma-Aldrich, Saint Louis, USA). RNA was isolated using the RNeasy PowerPlant Kit (Qiagen) using the protocol provided by the manufacturer. DNA co-isolated from the samples was removed with the RNase-Free DNase Set kit (Qiagen). RNA in the samples was quantified with the Qubit 2.0 fluorometer (Life Technologies, Carlsbad, USA) and RNA integrity was checked with Agilent TapeStation 4200 (Agilent Technologies).
5
RNA Extraction and Quality Assessment
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RNA was extracted using Invitrogen TRIzol Reagent (cat. #15596018), followed by genomic DNA removal and cleanup using Qiagen RNase-Free DNase Set kit (cat. #79254) and Qiagen Mini RNeasy kit (cat. #74104). An Agilent 2100 Bioanalyzer was used to assess the integrity of the RNA samples; only RNA samples having RNA Integrity Number score of 8–10 were sequenced.
6
Genomic DNA and RNA Isolation
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Both genomic DNA (gDNA) and total RNA were isolated from the total thyroid and thymus tissue using standard methods (QIAamp DNA Mini QIAcube Kit and RNeasy Mini Kit, QIAGEN, Hilden, Germany). An additional step of DNase I treatment was applied to the RNA samples (RNase-free DNase set kit, QIAGEN). To check for contaminating gDNA in the RNA samples, 100 ng of the total RNA were subjected to 45 cycles of PCR using specific primers for a 309 nt non-transcribed region of the CTLA4 promoter. Only samples free of DNA were used for subsequent experiments.
7
Ribosomal-Depleted RNA-Seq Profiling
8
Total RNA Extraction and Quality Assessment
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Total RNA was extracted from harvested cells using Invitrogen TRIzol® Reagent (cat#15596018), followed by genomic DNA removal and cleaning using Qiagen RNase-Free DNase Set kit (cat#79254) and Qiagen Mini RNeasy™ kit (cat#74104). Agilent 2100 Bioanalyzer was used to assess the integrity of the RNA samples. Only RNA samples having RNA Integrity Number between 8–10 were used.
9
Total RNA Extraction and Purification
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The extraction of total RNA from the samples was performed using Total RNA Purification Kit (Jena Bioscience Inc.) according to the manufacturer instruction. The impurity of the samples with genomic DNA was removed by RNase-Free DNase Set kit (Qiagen Inc.). The purity and concentration of the extracted RNAs were evaluated by NanoDrop® ND-1000 spectrophotometer. The extracted RNAs were then kept at -20°C until use.
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The extraction of total RNA from the samples was performed using Total RNA Purification Kit (Jena Bioscience Inc.) according to the manufacturer instruction. The impurity of the samples with genomic DNA was removed by RNase-Free DNase Set kit (Qiagen Inc.). The purity and concentration of the extracted RNAs were evaluated by NanoDrop® ND-1000 spectrophotometer. The extracted RNAs were then kept at -20°C until use.
10
Optimized RNA Extraction and Microarray Analysis
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