Total RNA was isolated from cells using an RNeasy® Mini Kit System (QIAGEN, Hilden, Germany). RNA quantity and purity were assessed with a Nanodrop Lite spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA (500 ng) was reverse-transcribed into cDNA using a PrimeScript™ RT Master Mix kit (TaKaRa Bio, Kusatsu, Japan). Real-time quantitative RT-PCR was performed with the SYBR Green fluorescent dye method using PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, Waltham, MA, USA) and a LightCycler® 480 II Real-Time PCR system (Roche, Basel, Switzerland). Primer pairs for each transcript (hNIS Forward 5′-GTAGAAGACCTCATCAAACCT-3′, hNIS Reverse 5′-GGAGCCCTGAAGGACACCTC-3′, GAPDH Forward 5′-CAACTACATGGTTTACATGTTCCAA-3′, GAPDH Reverse 5′-GCCAGTGGACTCCACGACGT-3′) were chosen with IDT SciTools (PrimerQuest™ program, IDT, Coralville, Iowa, USA. https://www.idtdna.com/SciTools , accessed on 9 September 2021) and “blasted” with NCBI (http://www.ncbi.nlm.nih.gov/BLAST/ , accessed on 9 September 2021). Amplification curves were read with LightCycler® 480 SW 1.5.1 software (Roche, Basel, Switzerland) using the comparative cycle threshold method. The steady-state level of mRNA for each gene of interest was normalized against the value for GAPDH mRNA.
Rneasy mini kit system
Manufactured by Qiagen
Sourced in Germany
The RNeasy Mini Kit is a silica-membrane-based system for the isolation and purification of total RNA from a variety of sample types, including animal cells, tissues, and bacteria. The kit utilizes a spin-column format and provides a simple and efficient method for the extraction of high-quality RNA, which can be used in various downstream applications such as RT-PCR, northern blotting, and microarray analysis.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler 420 2, by Roche (3 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop lite spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 sw 1, by Roche (1 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Primescript rt master mix kit, by Takara Bio (1 mentions)
Lightcycler 480 2 real time pcr system, by Roche (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rna free dnase, by Promega (1 mentions)
Moloney murine leukemia virus rt, by Promega (1 mentions)
Superscript 3 rt, by Thermo Fisher Scientific (1 mentions)
7 protocols using rneasy mini kit system
1
Quantitative RT-PCR Analysis of hNIS Expression
2
Quantification of CXCL13 and MCP-1 mRNA
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After organ retrieval, tissue was immediately fixed in RNAlater. Total mRNA was extracted using the RNeasy mini kit system (Qiagen, Hilden, Germany) and transcribed with Quiagen mini kits. For quantitative PCR (qPCR), 1 μg of DNase-treated total RNA was reverse transcribed using Superscript II Reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and qPCR was performed on a Lightcycler 420 II (Roche Diagnostics, Penzberg, Germany) using FastStart Sybr-Green. Gene-specific primers for CXCL13 (Primer-sequence: fwd-TCT GGA CCA AGA rev-TGA AGA AAG TT) and monocyte chemoattractant protein-1 (MCP-1; Mm_Ccl2_1_SG QuantiTect Primer Assay QT00167832) were used. Quantification was carried out using QGene software.
3
Quantitative PCR for Immune Gene Expression
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Total RNA was prepared using the RNeasy Mini Kit system (Qiagen). The quantity and quality of RNA was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific) cDNA copy of RNA isolated from cells was done according to the manufacturer's instruction (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems). Quantitative PCR was performed using primer/probes purchased from Applied Biosystems. GAPDH served as an internal reference standard. PCR was run on the 7500 Real-Time PCR System (Applied Biosystems). The following primer/probe sets were used: Tbx21 (Mm00450960_m1), Irf4 (Mm00516431_m1), Ebi3 (Mm00469294_m1), Il12a (Mm00434165_m1), Tgfb1 (Mm01178820_m1), Gata3 (Mm00484683_m1), Il10 (Mm00439614_m1), Blimp1 (Mm01187285_m1) and Gapdh (Mm99999915_g1).
4
Quantitative PCR for Inflammatory Genes
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Tissue sections were stored in RNA-later immediately after organ retrieval. Total RNA was extracted using the RNeasy mini kit system (Qiagen, Hilden, Germany) and transcribed using Superscript II Reverse transcriptase (Invitrogen). Quantitative (q) PCR was performed on Lightcycler 420 II (Roche Diagnostics, Penzberg, Germany) using FastStart Sybr-Green chemistry. Gene-specific primers for IL–6 (Quantitec QT00098875, Qiagen) and MCP–1 (Quantitec QT00167832, Qiagen) were used for the gene of interest and HPRT served as house keeping gene for normalization (Quantitec QT00166768, Qigaen). Quantification was carried out using qgene software.
5
Isolation and Analysis of Granulocyte and Monocyte RNA
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Total RNA of human granulocytes and monocytes was isolated using the RNeasy Mini Kit System (Qiagen, Venlo, The Netherlands), treated with RNA-free DNase (Promega, Madison, WI) and reverse transcribed using oligo(dT) primers and Moloney murine leukemia virus RT (Promega). Primers and RT-PCR conditions can be found in the online supplement (see Table S1, Supplemental Digital Content 1, at http://links.lww.com/SHK/A387 ). Data were analyzed using the comparative Ct method.
6
Semiquantitative RT-PCR Analysis of Cytokine Genes
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Total RNA was isolated from cell pellets using the RNeasy mini kit system (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instruction, and treated with DNase I to remove traces of contaminant DNA. The relative amount of each interest gene was determined by semiquantitative RT-PCR, as previously described [17 (link)]. Briefly, first strand cDNA synthesis utilized total RNA (1 μg) from each sample, oligo dT primers, and Super Script III RT (Invitrogen, Carlsbad, CA, USA). mRNA level of IL-6, IL-8 and 18S rRNA was determined by using following oligonucleotides as primers; IL-6 sense 5′-GACAGCCACTCACCTCTTCA-3′, anti-sense 5′-CATCTTTGGAAGGTTCAGGTTGT-3′, IL-8 sense 5′-CAGCCTTCCTGATTTCTGC-3′, anti-sense 5′-ACTTCTCCACAACCCTCTGC-3′, 18S rRNA sense 5′-ATCCTGCCAGTAGCATATGC-3′, anti-sense 5′-ACCCGGGTTGGTTTTGATCTG-3′. RT-PCR products were analyzed on 2% agarose gels, and bands were visualized by ethidium bromide staining. As an internal control for equal mRNA expression, 18S rRNA was used. The relative amount of RT-PCR products was expressed as fold increase relative to mock controls.
7
Quantitative PCR Analysis of Renal mRNA
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For mRNA work-up one part of the kidneys was fixed in RNAlater immediately. For total RNA extraction the RNeasy mini kit system (Qiagen, Hilden, Germany) was used and RNA was transcribed with Qiagen mini kits. For quantitative PCR (qPCR) 1 µg of DNase-treated total RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen) and qPCR was performed on a Lightcycler 420 II (Roche Diagnostics, Penzberg, Germany) using FastStart Sybr-Green chemistry. Gene-specific primers for MCP-1 (QT00167832) and IL-6 (QT00098875) were used. For normalization HPRT (QT00166768) was used as housekeeping gene. Quantification was carried out using qgene software.
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