Formalin fixation and paraffin embedding of ocular samples were performed immediately after tissue excision according to routine protocols, as previously described12 , 13 Following routine histological staining, each specimen's histological diagnosis was made by two experienced ophthalmic pathologists. Fifteen 4‐µm‐thick FFPE conjunctival sections were collected and stored in tubes before RNA extraction. RNA isolation from FFPE specimens was carried out as previously described.12 Briefly, total RNA was extracted from FFPE samples using the Quick‐RNA FFPE Kit (Zymo Research, Irvine, California). Following DNAse I digestion using the Baseline‐ZERO Kit (Epicentre, Madison, WI), the RNA concentration was quantified using the Qubit RNA HS Assay Kit on a Qubit Fluorometer (Life Technologies, Carlsbad, CA). RNA quality was determined via the RNA Pico Sensitivity Assay on a LabChip GXII Touch (PerkinElmer, Waltham, MA). RNA sequencing was performed using a massive analysis of complementary DNA ends (MACE), a 3′ RNA sequencing method, as previously described.12 The barcoded libraries comprising unique molecule identifiers were sequenced on the NextSeq 500 (Illumina) with 1× 75 bp. PCR bias was removed using unique molecular identifiers.
Baseline zero kit
Manufactured by Illumina
Sourced in United States
The Baseline-ZERO kit is a lab equipment product from Illumina. It is designed to provide a consistent and accurate baseline for researchers conducting various experiments and analyses. The core function of the Baseline-ZERO kit is to establish a reliable reference point for data collection and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Labchip gxii touch, by PerkinElmer (11 mentions)
Quick rna ffpe kit, by Zymo Research (11 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (11 mentions)
Qubit rna hs assay kit, by Thermo Fisher Scientific (10 mentions)
Nextseq 500, by Illumina (3 mentions)
Rna pico sensitivity assay, by PerkinElmer (2 mentions)
Hiseq 2500, by Illumina (1 mentions)
11 protocols using baseline zero kit
1
Ocular Tissue RNA Sequencing Protocol
2
RNA Extraction from FFPE Samples
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Total RNA was isolated from ten to fifteen formalin-fixed and paraffin-embedded (FFPE) 4-µm-thick sections from each specimen using the Quick-RNA FFPE Kit (Zymo Research, USA). Following a 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). The RNA quality was determined with the RNA Pico Sensitivity Assay on a LabChip GXII Touch (PerkinElmer, USA).
3
RNA Extraction from Corneal Samples
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RNA extraction from corneal samples was performed by a commercial provider (GenXPro, Frankfurt am Main, Germany) as previously described [9 (link)]. Briefly, total RNA was isolated using the Quick-RNA FFPE Kit (Zymo Research, Irvine, CA, USA). Following a DNAse I digestion using the Baseline-ZERO kit (Epicentre, Illumina, San Diego, CA, 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).
4
FFPE RNA Extraction and Quantification
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Fifteen FFPE sections of 4 μm thickness from each sample were stored in tubes prior to RNA extraction, as previously described [12 (link),13 (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, Illumina, San Diego, CA, USA), the RNA concentration was measured with the Qubit RNA HS Assay Kit (Life Technologies, Carlsbad, CA, USA) on a Qubit Fluorometer (Life Technologies, Carlsbad, CA, USA). The RNA quality was determined with the RNA Pico Sensitivity Assay (PerkinElmer, Waltham, MA, USA) on a LabChip GXII Touch (PerkinElmer, Waltham, MA, USA).
5
RNA Extraction from FFPE Samples
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Fifteen FFPE sections (4-mm thick) from each CNV membrane were collected and stored in tubes before RNA extraction. For the initial histopathologic evaluation, FFPE eyes of patients with ciliary body melanoma had been dissected by removing two scleral shells, leaving a central sclerocorneal ring segment. For the current study, the FFPE blocks were melted, and using a dissection microscope, the retina was carefully lifted off the RPE layer. Next, the RPE and choroid were cut laterally with a scalpel, and the central RPE/choroid complex, which easily detaches from the sclera in FFPE eyes, was retrieved and stored in low binding tubes until RNA extraction was performed. RNA isolation from FFPE specimens was performed as previously described. 19 Briefly, total RNA was extracted from FFPE samples by using the Quick-RNA FFPE Kit (Zymo Research, Irvine, CA). Following DNase I digestion using the Baseline-ZERO Kit (Epicentre Technologies, Madison, WI), the RNA concentration was quantified by using the Qubit RNA HS Assay Kit on a Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA). RNA quality was determined via the RNA Pico Sensitivity Assay on a LabChip GXII Touch (PerkinElmer, Waltham, MA). The fragment size of all RNA samples ranged between 120 and 150 bp.
6
FFPE RNA Sequencing and MACE Analysis
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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.
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.
7
RNA Extraction from FFPE Corneal Samples
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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).
8
FFPE RNA Extraction and MACE Sequencing
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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 .
9
RNA Isolation from FFPE Tissues
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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 described20 (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).
10
RNA Isolation from FFPE Samples
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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).
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