Quick rna ffpe kit

Total RNA was isolated from formalin-fixed and paraffin-embedded sections of all specimens using the Quick-RNA FFPE Kit (Zymo Research, USA). Following DNAse I digestion using the Baseline-ZERO kit (Epicentre, USA), the RNA concentration was measured with the Qubit RNA HS Assay Kit on a Qubit Fluorometer (Life Technologies, USA). RNA quality was determined with the RNA Pico Sensitivity Assay on a LabChip GXII Touch (PerkinElmer, USA).
Standard RNA-Seq libraries were prepared by GenXPro GmbH, as previously described [13 (link)]. The rRNA depletion and mRNA enrichment were performed via poly(A) selection and purification. All samples were sequenced strand-specific on the HiSeq2500 (Illumina, USA).
MACE libraries were constructed by GenXPro GmbH, as previously described [14 (link)]. Briefly, polyadenylated mRNA was isolated from 1 μg of total RNA. Twenty-eight barcoded libraries comprising unique molecule identifiers (UMIs) were sequenced on the NextSeq 500 (Illumina, USA) with 1 × 75 bp, followed by TrueQuant PCR bias elimination using UMIs. The sequencing data are available in the Gene Expression Omnibus Database under the accession number GSE149004.

RNA was extracted from fixed lung tissue in paraffin blocks using a Quick-RNA™ FFPE Kit (R1008, Zymo Research, Irvine, CA) following the manufacturer’s protocol [20 (link)]. Samples were deparaffinized, digested with proteinase K, and decrosslinked at 65 °C for 15 min. RNA lysis buffer was added and mixed with ethanol. The mixtures were transferred to spin columns to isolate total RNA.
RNA was extracted from cultured fibroblasts using a Direct-zol RNA MicroPrep kit (R2060; Zymo Research, Irvine, CA). Cells were washed with PBS and lysed in TRI reagent™, then purified using Direct-zol RNA columns. DNase I treatment was done in columns and RNA eluted in DNase/Rnase-free water.

RNA extraction from corneal samples was performed by a commercial provider (GenXPro, Frankfurt am Main, Germany) as previously described (Wolf et al., 2022b (link)). Briefly, total RNA was isolated from FFPE samples using the Quick-RNA FFPE Kit (Zymo Research, Irvine, CA, USA). Following a DNAse I digestion using the Baseline-ZERO kit (Epicentre, Madison, WI, USA), the RNA concentration was measured with the Qubit RNA HS Assay Kit on a Qubit Fluorometer (Life Technologies, Carlsbad, CA, USA). The RNA quality was determined with the RNA Pico Sensitivity Assay on a LabChip GXII Touch (PerkinElmer, Waltham, MA, USA).

Total RNA was isolated from formalin-fixed and paraffin-embedded (FFPE) sections of all specimens using the Quick-RNA FFPE Kit (Zymo Research, USA). Following a DNAse I digestion using the Baseline-ZERO kit (Epicenter, USA), the RNA concentration was measured with the Qubit RNA HS Assay Kit on a Qubit Fluorometer (Life Technologies, USA). The RNA quality was determined with the RNA Pico Sensitivity Assay on a LabChip GXII Touch (PerkinElmer, USA). The fragment size of all RNA samples ranged between 120 and 150 bp. The preparation of massive analysis of cDNA ends (MACE) libraries was carried out using 1 μg of total RNA, as previously described (Zajac et al., 2015 (link)). The barcoded libraries (four CNV membranes and four control samples) were sequenced simultaneously on the NextSeq 500 (Illumina, USA) with 1 × 75 bp. Data analysis was conducted as described above with the following modifications: Reads were mapped to the human reference genome (hg38, Galaxy built-in reference genome) with RNA STAR Galaxy Version 2.6.0b-2 6 (default parameters) using the Gencode annotation file (Gencode 31, release June 2019, downloaded on 08/05/2019, https://www.gencodegenes.org/human/releases.html). The sequencing data are available in the Gene Expression Omnibus Database under the accession number GSE146887.

After melting the paraffin block, the pterygium, as well as the control FFPE samples were stored in tubes until RNA isolation, which was performed as previously described (21 (link), 22 (link)). Briefly, total RNA was isolated from FFPE samples using the Quick-RNA FFPE Kit (Zymo Research). Following a DNAse I digestion using the Baseline-ZERO kit (Epicentre), the RNA concentration was measured with the Qubit RNA HS Assay Kit on a Qubit Fluorometer (Life Technologies). The RNA quality was determined with the RNA Pico Sensitivity Assay on a LabChip GXII Touch (PerkinElmer).

After melting the block, tumor-free tissue areas were removed and the tumor, as well as the control FFPE samples were stored in tubes until RNA isolation, which was performed as previously described^{20 (link),70 (link)}. Briefly, total RNA was isolated from FFPE samples using the Quick-RNA FFPE Kit (Zymo Research). Following a DNAse I digestion using the Baseline-ZERO kit (Epicentre), the RNA concentration was measured with the Qubit RNA HS Assay Kit on a Qubit Fluorometer (Life Technologies). The RNA quality was determined with the RNA Pico Sensitivity Assay on a LabChip GXII Touch (PerkinElmer).

DNA was isolated from blood and saliva using Gentra Purgene Blood Kit (Qiagen, Germantown, MD), or from frozen lung tissue using DNeasy Blood and Tissue Kit (Qiagen). RNA from frozen lung or FFPE lung tissues was extracted using miRNeasy Mini Kit (Qiagen) or Quick-RNA FFPE Kit (Zymo Research, Irvine, CA), respectively. DNA and RNA from cultured IMR-90 cells was isolated using DNaesy Blood and Tissue Kit (Qiagen) and miRNeasy Mini Kit (Qiagen), respectively.