Mag Jet RNA Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for the extraction of total RNA. The RNA’s quality was estimated by electrophoresis in the presence of 1 μg/mL ethidium bromide (2% agarose gel in Tris/Borate/EDTA buffer). The concentration of the extracted RNA was determined with NanoDrop 1000c spectrophotometer. RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for reverse transcription of total RNA.
Mag jet rna kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Mag Jet RNA Kit is a laboratory equipment product designed for the rapid and efficient extraction of RNA from a variety of sample types. The kit utilizes a magnetic bead-based technology to capture, wash, and elute RNA, providing a convenient and reliable method for RNA purification.
Automatically generated - may contain errors
Lab products found in correlation
Revertaid h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (22 mentions)
Ncounter gene expression assay, by NanoString (7 mentions)
Nsolver software, by NanoString (6 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (5 mentions)
2100 bioanalyzer instrument, by Agilent Technologies (4 mentions)
Kingfisher duo machine, by Thermo Fisher Scientific (4 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Kingfisher duo rna extraction machine, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
High capacity cdna transcription kit, by Thermo Fisher Scientific (1 mentions)
Cfx384 real time system, by Bio-Rad (1 mentions)
C1000 thermal cycler, by Bio-Rad (1 mentions)
Ssofast evagreen supermix, by Bio-Rad (1 mentions)
Recombinant shrimp alkaline phosphatase, by New England Biolabs (1 mentions)
Truncated t4 rna ligase 2, by New England Biolabs (1 mentions)
Oligo d t 25 magnetic beads, by New England Biolabs (1 mentions)
Trueseq small rna protocol, by Illumina (1 mentions)
T4 rna ligase 1, by New England Biolabs (1 mentions)
T4 polynucleotide kinase, by New England Biolabs (1 mentions)
24 protocols using mag jet rna kit
1
Total RNA Extraction and cDNA Synthesis
2
RNA Extraction and Reverse Transcription
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As in our previous works39 (link),40 (link) Mag Jet RNA Kit (Thermo Fisher Scientific, USA) was used for the extraction of total RNA. The RNA quality was estimated by electrophoresis in the presence of 1 μg/ml ethidium bromide (2% agarose gel in Tris/Borate/EDTA buffer). The concentration of the extracted RNA was determined with NanoDrop 1000c spectrophotometer. RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA) was used for reverse transcription of total RNA.
3
RNA Extraction and qRT-PCR Protocol
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Total RNA was extracted using MagJet RNA kit (Thermo, K2731), and reverse transcribed using the High Capacity cDNA Transcription kit (Applied Biosystems, 4368814) following the manufacturer’s instructions. Real-Time PCR was performed using the Ssofast Evagreen Supermix (BioRad, 1725201) in a C1000 Thermal Cycler with CFX384 Real-Time System (BioRad) using the following cycling conditions: initial denaturation of 30s at 95C followed by 40 cycles of 5s at 95C and 5s at 60C. Cebpa expression relative to Hprt was computed as , where and are the threshold cycles for Cebpa and Hprt respectively. The following primers were used:
The data are provided inS1 Dataset .
The data are provided in
4
Transcription Factor Expression Analysis
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RNA was extracted separately from snap-frozen DD cords and nodules from the original cohort of 6 patients used for DAB immunohistochemical staining, and was used for NanoString nCounter Gene Expression Assay (Nanostring Technologies, Seattle, Wash.). Total RNA was extracted using the MagJET RNA kit (Thermo Fisher Scientific) with the protocol adapted for tissue, and run on a KingFisher Duo machine (Thermo Fisher Scientific). RNA samples were then quantitated on a Qubit 2.0 fluorometer (Life Technologies) and were subjected to RNA integrity analysis using the 2100 Bioanalyzer Instrument (Agilent Technologies, Santa Clara, Calif.). Samples then underwent NanoString nCounter Gene Expression Assay performed by New Zealand Genomics (Dunedin, New Zealand) according to the manufacturer’s protocol. Probes for the genes encoding NANOG (NM_024865.2), SALL4 (NM_020436.3), SOX2 (NM_003106.2), OCT4 (NM_002701.4), and STAT3 (NM_139276.2) and the housekeeping gene GUSB (NM_000181.1) were designed and manufactured by NanoString Technologies. Raw data was analyzed using nSolver software (NanoString Technologies) using standard settings and normalized against the housekeeping gene.
5
Quantifying Stem Cell Markers in MDLSCC
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Total RNA was isolated and quantified as previously described (31 (link)) from six snap-frozen MDLSCC samples from the original cohort of 10 patients used for DAB IHC staining. They were subjected to the NanoString nCounter gene expression assay (NanoString Technologies, Seattle, WA, USA) by New Zealand Genomics (Dunedin, New Zealand). Briefly, total RNA was extracted using the MagJET RNA kit (Thermo Fisher Scientific) with the protocol adapted for tissue and run on a KingFisher Duo machine (Thermo Fisher Scientific). RNA samples were then quantitated on a Qubit® 2.0 fluorometer (Thermo Fisher Scientific) and were subject to RNA integrity analysis via the 2100 Bioanalyzer Instrument (Agilent Technologies). Probes for the genes encoding CD44 (NM_001001392.1), NANOG (NM_024865.2), OCT4 (NM_002701.4), STAT3 (NM_139276.2), and the housekeeping genes glucuronidase beta (GUSB) (NM_000181.1), clathrin heavy chain (CLTC) (NM_4859.2), and hypoxanthine phosphoribosyltransferase 1 (NM_000194.1) were designed and manufactured by NanoString Technologies. Raw data were analyzed using nSolver™ software (NanoString Technologies) using standard settings and normalized against the housekeeping gene.
6
Quantification of Pluripotency Genes in CAML
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RNA was extracted from six snap-frozen samples of CAML from the same cohort of nine patients used for DAB IHC staining, was analyzed using NanoString nCounter™ Gene Expression Assay (NanoString Technologies, Seattle, WA, USA). Total RNA was extracted using the MagJET RNA kit (Thermo Fisher Scientific) with the protocol adapted for tissue, and run on a KingFisher Duo machine (Thermo Fisher Scientific). The RNA samples were then quantitated on a Qubit® 2.0 fluorometer (Invitrogen, Life Technologies) and were subject to RNA integrity analysis via the 2100 Bioanalyzer Instrument (Agilent Technologies). The samples then underwent NanoString nCounter gene expression assay performed by New Zealand Genomics (Dunedin, New Zealand) according to the manufacturer’s protocol. Probes for the genes encoding OCT4 (POU5F1, NM_002701.4), SOX2 (NM_003106.2), NANOG (NM_024865.2), KLF4 (NM_004235.4), c-Myc (NM_002467.3), and the housekeeping gene GUSB (NM_000181.1) were designed and synthesized by NanoString Technologies. Raw data were analyzed using nSolver™ software (NanoString Technologies) using standard settings and normalized against the housekeeping gene.
7
Strand-specific RNA-seq library preparation
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Total RNA extracted using the MagJET RNA kit (Thermo Scientific) was first checked for integrity on an Agilent Bioanalyzer 2100; samples with RNA integrity number (RIN) >9.0 were used for subsequent processing. Total RNA was subjected to two rounds of poly(A) selection using oligo-d(T)25 magnetic beads (New England Biolabs). A single-read cDNA library was prepared following the Illumina TrueSeq small RNA protocol for strand-specific RNA-seq with minor modifications (Hoque et al. 2013 (link)). Briefly, poly(A)+ RNA was fragmented in an alkaline buffer (NaHCO3 at pH 9.3) for 2 min at 94°C followed by dephosphorylation with recombinant shrimp alkaline phosphatase (New England Biolabs) and then phosphorylation with T4 polynucleotide kinase (New England Biolabs). After addition of 3′ adapter (5′ adenylated) and 5′ adapter using truncated T4 RNA ligase II (New England Biolabs) and T4 RNA ligase I (New England Biolabs), respectively, RNA was reverse-transcribed using 3′ adapter-specific primer. cDNA was then amplified by PCR for 15 cycles with a universal forward primer and a reverse primer with bar code. The cDNA libraries were purified from an 8% polyacrylamide gel and quantified on an Agilent Bioanalyzer.
8
Renin-Angiotensin System Gene Expression
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Snap-frozen DD cord (n = 5) and DD nodule (n = 5) samples from 5 patients from the same cohort included for DAB IHC staining were used to isolate total RNA. RNA was extracted using the MagJET RNA Kit and KingFisher Duo (ThermoFisher Scientific) protocol and quantitated by the NanoDrop 2000 Spectrophotometer (Thermo Scientific) and Qubit (Thermo Scientific). mRNA was assayed by New Zealand Genomics Ltd (Dunedin, NZ), using the NanoString nCounter Gene Expression Assay (NanoString Technologies, Seattle, Wash.). Probes for the genes were designed and synthesized by NanoString Technologies and are PRR (ATP6AP2, NM_005765.2), ACE (CD143, NM_000789.2), ATIIR1 (AGTR1, NM_000685.3), and ATIIR2 (AGTR2, NM_000686.3). Raw data were analyzed by nSolver software (NanoString Technologies) using standard settings and were normalized against the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
9
RNA Extraction and cDNA Synthesis
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A MagJET RNA Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for the extraction of total RNA. The RNA quality was estimated by electrophoresis in the presence of 1 μg/mL ethidium bromide (2% agarose gel in Tris/borate/EDTA buffer). The concentration of the extracted RNA was determined with a NanoDrop 1000c spectrophotometer. A RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA) was used for reverse transcription of total RNA.
10
Quantitative Gene Expression Analysis of Stem Cell Markers
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RNA was extracted from five snap-frozen MDBMSCC samples from the same cohort of patients used for DAB IHC staining and was used for NanoString nCounter™ Gene Expression Assay (Nanostring Technologies, Seattle, WA, USA). Total RNA was extracted using the MagJET RNA kit (Thermo Fisher Scientific) with the protocol adapted for tissue and run on a KingFisher Duo machine (Thermo Fisher Scientific). RNA samples were then quantitated on a Qubit® 2.0 fluorometer (Invitrogen, Life Technologies) and were subject to RNA integrity analysis via the 2100 Bioanalyzer Instrument (Agilent Technologies). The samples then underwent NanoString nCounter gene expression assay performed by New Zealand Genomics (Dunedin, New Zealand) according to the manufacturer’s protocol. Probes for the genes encoding NANOG (NM_024865.2), SALL4 (NM_020436.3) SOX2 (NM_003106.2), OCT4 (NM_002701.4), CD44 (NM_001001392.1), and STAT3 (NM_139276.2) and the housekeeping gene GUSB (NM_000181.1) were designed and manufactured by NanoString Technologies. Raw data were analyzed using nSolver™ software (NanoString Technologies) using standard settings and normalized against the housekeeping gene.
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