Cells were treated as mentioned and after 18 hours total RNA was extracted using Zymo Research Quick-RNATM MiniPrep kit according to the manufacturer's instructions. RNA was treated with DNase-1 prior to reverse transcription. Real-time Quantitative PCR was performed using Power Sybr®Green (Applied Biosciences) on a Bio-Rad CFX 96 RealTime System C100 Thermal Cycler. Relative gene expression levels were determined by normalization to β-actin level using the ΔΔCt method. Primers sequences are available upon request.
Quick rnatm miniprep kit
Manufactured by Zymo Research
Sourced in United States
The Quick-RNA™ MiniPrep kit is a rapid and efficient tool for the isolation of high-quality RNA from a variety of sample types. The kit utilizes a proprietary technology to ensure the effective removal of genomic DNA, proteins, and other contaminants, resulting in pure RNA suitable for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (4 mentions)
Nextera dna flex library preparation kit, by Illumina (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Sybr fast qpcr kit, by Roche (2 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Stratagene mx3005p real time pcr detection system, by Agilent Technologies (2 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
M mlv reverse transcriptase, by Promega (2 mentions)
Sybr green supermix, by Thermo Fisher Scientific (2 mentions)
Cfx 96 real time system c100 thermal cycler, by Bio-Rad (1 mentions)
Miniseq, by Illumina (1 mentions)
Agilent high sensitivity dna kit, by Agilent Technologies (1 mentions)
Fragment analyzer system, by Agilent Technologies (1 mentions)
Ampure xp, by Beckman Coulter (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Maxima h minus double stranded cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Lunascript rt supermix kit, by New England Biolabs (1 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (1 mentions)
34 protocols using quick rnatm miniprep kit
1
Quantitative PCR Analysis of Gene Expression
2
RNA Extraction and Sequencing for Viral Genomics
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RNA extraction was performed using the Quick-RNATM MiniPrep kit (Zymo Research, Irvine, CA, USA) and 200 µL of purified viral particles. Reverse transcription and double-stranded synthesis were carried out using the Maxima H Minus Double-Stranded cDNA Synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA) and 13 µL of extracted RNA. Nextera™ DNA Flex Library Preparation kit (Illumina, San Diego, CA, USA) was used from 1 ng of double-strand cDNA. The libraries were purified with AMPure XP (Beckman Coulter, Indianapolis, IN, USA) and quantified with the Qubit dsDNA HS assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The library’s quality and length were assessed with the Agilent high-sensitivity DNA kit (Agilent, Santa Clara, CA, USA) using a Fragment Analyzer™ System (Advanced Analytical Technologies, Inc., Heidelberg, Germany, Europe). Sequencing was performed on the MiniSeq and MiSeq platforms (Illumina, San Diego, CA, USA).
3
RNA Extraction and cDNA Synthesis
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After being submitted to the initial treatment with or without compounds and incubated at 37°C for 5 h with shaking, total RNA was extracted using the Quick-RNATM Miniprep kit (Zymo Research, Irvine, USA). The Turbo DNA-freeTM kit (Invitrogen, Waltham, USA) was then used to remove contaminating DNA from RNA preparations, and DNase and divalent cations from the samples. cDNA synthesis was done using the LunaScript® RT SuperMix kit (New England Biolabs, Ipswich, USA). All experiments were performed following the manufacturers’ instructions. Finally, cDNA samples concentrations were measured using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, USA).
4
Quantification of SMURF2 Expression
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Total RNA was extracted by Quick-RNATM MiniPrep Kit (Zymo Research Corporation, Irvine, CA, USA). Then 1 μg of extracted RNA was used for cDNA synthesis with random hexamers provided in RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) followed by SYBR Green-based qPCR reaction (SYBR® FAST qPCR Kit, Kapa Biosystems, Inc., Wilmington, MA, USA). The cycling conditions were as follows: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 sec and 60 °C for 1 min. The end-point used in the real-time quantification was calculated by the StepOne software (v2.2.2, Applied Biosystmes, Carlsbad, CA, USA, 2011), and the threshold cycle number (Ct value) for each analyzed sample was calculated. The primer sets used in this study were listed as followed:
SMURF2
Forward: 5’-TAGCCCTGGCAGACCTCTTA-3’
Reverse: 5’- AATACACCTGGCCTTGTTGC-3’
MRPL19 (internal control)
Forward: 5’- GGGATTTGCATTCAGAGATCAG-3’
Reverse: 5’- GGAAGGGCATCTCGTAAG-3’
qPCR data were analyzed as previous described [35 (link)].
5
Transcriptional Profiling of Hematopoietic Cells
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RNA was isolated from cells using the Quick-RNA TM Miniprep Kit (Zymo Research) on days 3 and 6 or 7 post-transduction (for ML-2 and K562, respectively; the late time point was selected based on depletion kinetics, see Fig. 2c). PolyA-enriched total cellular RNA sequencing was performed by Novogene Company, Ltd. Differential expression analysis was conducted in R using DESeq2 26 (Bioconductor). Gene sets from MSigDB v7.2 (H1, C2, C3, C6), custom hematopoietic 16 (link) and chromosome 15 gene sets, and PAF1c-knockout expression signatures 27 were checked for enrichment in the Broad GSEA software 28 . Custom positional gene sets were generated by walking a 1 Mb or 5 Mb window along chromosome 15. Gene ontology analysis was performed using the DAVID 29 online functional annotation tool (https://david.ncifcrf.gov/summary.jsp).
6
Fibroblast DNA and RNA Extraction and Sequencing
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Genomic DNA from fibroblasts was extracted using a Quick-DNATM Miniprep Plus Kit, Zymo Research, Irvine, CA, USA, according to manufacturer’s instructions, followed by PCR to amplify desired regions on each gene. Gel extraction of the PCR product was performed with a ZymocleanTM Gel DNA recovery kit, Zymo Research, followed by Sanger sequencing.
Total RNA was isolated from fibroblasts using a Quick-RNATM MiniPrep kit, Zymo Research, and cDNA was synthesized using SuperScript III First-Strand Synthesis System, Thermo Fisher Scientific, Waltham, MA, USA, according to manufacturer’s instructions. cDNA was used for RT-PCR, and gel extraction of the PCR products was performed with the ZymocleanTM Gel DNA recovery kit, Zymo Research, followed by Sanger sequencing.
Total RNA was isolated from fibroblasts using a Quick-RNATM MiniPrep kit, Zymo Research, and cDNA was synthesized using SuperScript III First-Strand Synthesis System, Thermo Fisher Scientific, Waltham, MA, USA, according to manufacturer’s instructions. cDNA was used for RT-PCR, and gel extraction of the PCR products was performed with the ZymocleanTM Gel DNA recovery kit, Zymo Research, followed by Sanger sequencing.
7
Total RNA Extraction and Validation
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Total cellular RNA was extracted according to the manufacturer’s instructions by using a Quick-RNATM MiniPrep kit (Zymo Research, CA, USA). RNA was quantified based on the absorbance ratio at 260/280 nm wavelength using a Nanodrop 2000c Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The integrity of the 28S and 18S ribosomal RNAs was confirmed by electrophoresis on a 1% agarose gel followed by ethidium bromide staining, and the 28S/18S ratio was larger than 2.
8
Quantitative Analysis of miR-494-3p and Bmi1 Expression
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Total RNA was extracted by Quick-RNATM MiniPrep Kit (Zymo Research Corporation, Irvine, CA, USA). For miR-494-3p detection, 100 ng extracted RNA was used for complementary DNA (cDNA) synthesis with RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc.) and a specific RT primer (RiboBio Co., Ltd.). qPCR was performed on an ABI StepOnePlus™ Real-Time PCR System (Applied Biosystems, Life Technologies Corp., Carlsbad, CA, USA) by SYBR Green reagent (SYBR® FAST qPCR Kit, Kapa Biosystems, Inc., Wilmington, MA, USA) with specific primers purchased from RiboBio Co., Ltd. and analyzed with the StepOne software (Applied Biosystems). For Bmi1 detection, 1 μg of extracted RNA was used for cDNA synthesis with random hexamers provided in RevertAid First Strand cDNA Synthesis Kit followed by SYBR Green based qPCR reaction. The primer sequences were listed as followed:
Bmi1
Forward: 5′-AATCCCCACCTGATGTGTGT-3′
Reverse: 5′-GCTGGTCTCCAGGTAACGAA-3′
MRPL19 (internal control)
Forward: 5′-GGGATTTGCATTCAGAGATCAG-3′
Reverse: 5′-GGAAGGGCATCTCGTAAG-3′
qPCR data were analyzed as previous described [28 (link)].
9
Transcriptional Profiling of Hematopoietic Cells
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RNA was isolated from cells using the Quick-RNA TM Miniprep Kit (Zymo Research) on days 3 and 6 or 7 post-transduction (for ML-2 and K562, respectively; the late time point was selected based on depletion kinetics, see Fig. 2c). PolyA-enriched total cellular RNA sequencing was performed by Novogene Company, Ltd. Differential expression analysis was conducted in R using DESeq2 26 (Bioconductor). Gene sets from MSigDB v7.2 (H1, C2, C3, C6), custom hematopoietic 16 (link) and chromosome 15 gene sets, and PAF1c-knockout expression signatures 27 were checked for enrichment in the Broad GSEA software 28 . Custom positional gene sets were generated by walking a 1 Mb or 5 Mb window along chromosome 15. Gene ontology analysis was performed using the DAVID 29 online functional annotation tool (https://david.ncifcrf.gov/summary.jsp).
10
Quantifying mRNA Expression in Cultured Cells
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Total RNA was isolated from cultured cells using the Quick-RNATM MiniPrep kit (Zymo Research, Orange, California, USA), followed by qRT-PCR with the Power SYBR Green RNA-to-CT 1-Step Kit (Applied Biosystems, Foster City, California, USA) 48 h after transfection. Specifically, primers purchased from QIAGEN (Hilden, Germany) were used to perform qRT-PCR using the Stratagene Mx3005P Real-Time PCR detection system (Agilent Technologies, Santa Clara, California, USA). All experiments were normalized with β-actin as an internal control.
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