Cells were harvested, washed with 1xPBS and lysed in Qiazol Lysis regent (Qiagen). Total RNA was isolated using the miRNeasy kit (Qiagen). RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription kit (Thermo Fisher) and gene expression was measured using the following Taqman gene expression assays (Thermo Fisher): KIT assay Hs00174029_m1, GYPA assay Hs00266777_m1, SLC41A1 assay Hs00998197_m1, SLC22A4 assay Hs01548718_m1, HBG2/HBG1 assay Hs00361131_g1, 18s assay Hs99999901_s1. Fold change of expression was determined by comparative dCT methods using ribosomal18s gene as the endogenous control. Transcript copy number was determined by relative standard curve generated from oligonucleotides and normalized to 18s transcript copy number.
Qiazol lysis regent
Manufactured by Qiagen
Qiazol Lysis Reagent is a monophasic solution of phenol, guanidine isothiocyanate, and other proprietary components designed for the isolation of total RNA from a variety of biological samples, including cells, tissues, and microorganisms.
Automatically generated - may contain errors
Lab products found in correlation
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Mirneasy kit, by Qiagen (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Bullet blender, by Qiagen (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Rneasy plus universal mini kit, by Qiagen (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Nebnext rrna depletion kit v2, by New England Biolabs (1 mentions)
2 protocols using qiazol lysis regent
1
RNA Extraction and Expression Analysis of Key Genes
2
RNA Extraction and Sequencing from Rodent Tissues
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RNA was prepared as previously described27 (link). In summary, small sections of cotton rat lung (lingular lobe), large intestines (~ 20 mm colon, flushed with sterile PBS), spleen, kidney, and heart were homogenized using a NextAdvance Bullet Blender® with RED RINO™ tubes containing 3.2 mm stainless steel beads (SSB32) and 2 × volume of QIAzol® Lysis Regent (Qiagen, cat. no. 79306) at max speed for 3 min. RNA was extracted from homogenates using RNeasy® Plus Universal Mini kit (Qiagen, cat. No. 73404) via additional QIAzol® (total volume 900uL), gDNA eliminator, and chloroform with column DNase digestion as recommend by the manufacturer’s protocol. RNA quality was measured using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). Host rRNA was removed using the NEBNext rRNA Depletion Kit v2 (Cat no: NEB E7400X). The cDNA libraries were prepared using 1 μg total RNA from each sample using the NEBNext Ultra II RNA Library Prep Kit for Illumina (Cat no: NEB E7805L) following the manufacturers protocol for intact RNA (RIN > 7), AMPure XP Beads for cleanup steps (Beckman, cat. No. A63881), and NEBNext Multiplex Oligos for Illumina (Set 1, cat no: E7600S) for pooling samples. Sequencing was performed using Illumina NovaSeq6000 2 × 150 bp sequencing at the Vanderbilt Technologies for Applied Genomics (VANTAGE) core facility.
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