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Int. J. Mol. Sci. 2020, 21, 2370 15 of 20 4.7. Laser Microdissection of Photoreceptors The cultivated, unfixed and snap frozen eyes were sectioned vertically at 25-μm thickness. Slides were fixed in ice cold 70% ethanol for 30 sec, washed shortly in ice cold DEPC-treated water, dehydrated twice for 1 min in ice cold 100% ethanol and air dried for 5–10 min. To cut out the photoreceptor layer (ONL, IS, OS), a laser dissection microscope was used (PALM Carl Zeiss with PALM Robo software). Only the central area of the retina (2/3 of the total length) was dissected and collected from each section, in total six–eight sections per sample. 4.8. RNA Isolation RNA isolation of the dissected photoreceptors was done using the RNeasy Micro plus (Qiagen, Hilden, Germany) Kit according manufacturer’s instruction. The collected tissue from laser microdissection was dissolved in RLT buffer containing β-Mercaptoethanol. The integrity and concentration of isolated RNA were assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). 4.9. Sequencing and Bioinformatic Analysis Complete cDNA was synthesized from 5 μl total RNA using the SmartScribe reverse transcriptase (Takara Bio, Kusatsu, Japan) with a universally tailed poly-dT primer and a template switching oligo followed by amplification for 12 cycles with the Advantage 2 DNA Polymerase (Takara Bio, Kusatsu, Japan). After ultrasonic shearing (Covaris S2, Woburn, Massachusetts, USA), amplified cDNA samples were subjected to standard Illumina fragment library preparation using the NEBnext Ultra DNA library preparation chemistry (New England Biolabs, Ipswich, MA, USA). In brief, cDNA fragments were end-repaired, A-tailed and ligated to indexed Illumina Truseq adapters. Resulting libraries were PCR-amplified for 15 cycles using universal primers, purified using XP beads (Beckman Coulter, Brea, CA, USA) and then quantified with qPCR (KAPA Biosystems, Basel, Switzerland). Final libraries were equimolarly pooled and subjected to 75-bp-single-end sequencing on the Illumina HiSeq 2500 platform (Illumina Inc., San Diego, CA, USA), resulting in ~25–32 mio reads. Reads were mapped to the mouse genome (version mm10) with GSNAP (PMID:20147302; v2016-06-30) and splice sites from Ensembl (version 81) as support. RNA-seq data quality was assessed with RNA-SeQC (PMID:22539670; v1.1.8). Uniquely mapped reads served as an input for obtaining gene counts with featureCounts (PMID:24227677; v1.5.0) and Ensembl gene annotations (version 81). Normalization for library size and identification of differentially expressed genes was done with the R package DESeq2 (PMID:2551628; v1.12.13). To reduce the impact of sex-specific variance in gene expression, two male samples were included. Additional sources of unwanted variation in the data were modeled with the R package sva (PMID:22257669; v3.20.0) and included in the experiment design. DESeq2 p-values were adjusted for multiple testing (Benjamini-Hochberg) and genes with an adjusted p-value < 0.1 were considered as differentially expressed. The heat map of differentially expressed genes was generated using http://heatmapper.ca/. 4.10. In-situ Hybridization For ISH we used two PCR-generated probes for cryaa and cryab. Primers used for probe generation are listed in the supplement, Table S2. PCR fragments were cloned into TOPO®vector. Probes were linearized with HindIII and transcribed with T7 using DIG RNA Labeling Kit SP6/T7 (Roche, Basel, Switzerland). Control sense probes were linearized with XhoI and transcribed with SP6 using DIG RNA Labeling Kit SP6/T7 (Roche 11 175 025 910, Basel, Switzerland). PFA fixed cryosections (14 μm) were washed two times with PBST (DEPC treated 1xPBS + 0.2% Tween 20) and pre-hybridized with hybridization solution (50% formamide, 5× SSC, 1× Denharts, 0.2 mg/mL Yeast RNA, 0.1 mg/mL Heparin, 0.1% CHAPS, 0.01 M EDTA, 0.4% Tween-20), for 1 h at 70 ◦C. Probes were diluted in hybridization solution, added to the slides and incubated at 70 ◦C over night. Unhybridized RNAPDF Image | Photobiomodulation Mediates Neuroprotection against Retinal
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