Nucleospin rna kit

Total RNA was extracted from 30 MT pairs per genotype from female flies per genotype using NucleoSpin RNA kit (Fisher Scientific, # NC9581114). cDNAs were synthesized using the iScript cDNA synthesis kit (Bio-Rad, #1708896) in a 20 μl reaction mixture containing 500 ng total RNA. Quantitative real-time RT-PCR (RT-qPCR) assays were performed using iQ SYBR Green Supermix (Bio-Rad, #1708880) on a CFX96 Real-Time PCR Detection System (Bio-Rad). RT-qPCR reactions were carried out with gene-specific primers (Supplementary Data 3). A 5-fold serial dilution of pooled cDNA was used as the template for standard curves. Quantitative mRNA measurements were performed in three independent biological replicates and three mechanical replicates, and data were normalized to the amount of Dmrp49 mRNA.

RNA was isolated from tissues using the RNeasy kit (Qiagen) according to the manufactures instructions and total RNA from cultured cells was isolated using a Nucleospin RNA kit (Fisher Scientific). cDNA synthesis was performed using 1 μg of total RNA isolated from tissues or from cells and mastermix containing a blend of random primer (Roche), RT enzyme (Roche) and dNTP (Roche)and real-time PCR amplification was performed as described previously [13 (link)] using a LightCycler^® 480 Real-Time PCR System (Roche, Dublin, Ireland). The results were normalised to the β actin gene and relative expression was calculated based on the 2^-ΔΔCt method as previously described [17 (link)]. All gene-specific primer sequences used in the study are listed in S1 and S2 Tables.

RNA was isolated (Machery Nagel NucleoSpin RNA Kit, Dueren, Germany) and quality was assessed with NanoDrop (Thermo Fisher Scientific Inc., Waltham, MA, USA) and Bioanalyser measurements (Agilent, Santa Clara, CA, USA). The RNA sequencing data quality was assessed using FastQC (V0.11.4, Babraham Institute, Cambridge, UK) [53 ] before aligning reads with STAR (V2.5.4b) [54 (link)] against the Ensembl H. sapiens genome V91. The alignment quality was analyzed using samtools (V1.1) [55 (link)]. For all genes, normalized read counts were obtained using GenomicAlignments (V1.14.2) and DESeq2 (V1.18.1) [56 (link)]. Transcripts covered with <50 reads were excluded from subsequent analyses. The significance thresholds were set to |log2 fold-change| ≥ 1 and BH-adjusted p ≤ 0.01. To minimize sample variations, surrogate variable analysis (sva, V3.26.0) was used [57 (link)]. nRPKMs (normalized reads per kilobase per million total reads) were calculated from raw counts from DESeq2 [58 (link)]. Using gene sets provided by Andersson et al. and Stam et al. [59 (link),60 (link)], gene set enrichments were determined with GSEA (V3.0) [61 (link),62 (link)]. RNA-seq data were deposited in NCBI’s Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE128342.