Flashtag biotin hsr rna labeling kit

The miRNA expression profiling was carried out using Affymetrix platform-based microarrays with GeneChip™ miRNA 4.1 Array Strip (ThermoFisher Scientific, Waltham, MA, USA). The technical procedure that was followed was previously described in detail elsewhere [110 (link),111 (link)]. Each microarray was designed following the miRBase Release 20 database, which included complementary probes for 2578 human mature miRNA, 1996 human snoRNA, CDBox RNA, H/ACA Box RNA, scaRNA, and 2025 human pre-miRNA. miRNA was prepared for the hybridization step with the FlashTagTM Biotin HSR RNA Labeling Kit (ThermoFisher Scientific, Waltham, MA, USA). During this process, 150 ng of miRNA was subjected to the poly(A) tailing and biotin ligation in accordance with the producer’s protocol. Biotin-labeled miRNA were then hybridized to GeneChip™ miRNA 4.1 Array Strip (20 h, 48 °C). Afterwards, the microarrays were washed and stained following the technical protocol and using the Affymetrix GeneAtlas Fluidics Station (Affymetrix, Santa Clara, CA, USA). Next, the array strips were scanned using GeneAtlas System Imaging Station (Thermo Fisher Scientific, Waltham, MA, USA).

miRNA expression profiling was performed using the microarray approach with Applied BiosystemsTM miRNA 3.1 Array Strip (ThermoFisher Scientific, Waltham, MA, USA). The detailed technical procedure was described earlier [116 (link),117 (link)]. Each microarray was designed in accordance with the miRBase Release 17 database, including complementary probes for: 1733 human mature miRNA, 2216 human snoRNA, CDBox RNA, H/ACA Box RNA, and scaRNA, 1658 human pre-miRNA. The full procedure for preparing miRNA for hybridization was performed using the FlashTagTM Biotin HSR RNA Labeling Kit (ThermoFisher Scientific, Waltham, MA, USA). Then, 150 ng of previously isolated miRNA was subjected to the poly(A) tailing and biotin ligation procedure, according to the manufacturer’s protocol. Biotin-labeled miRNA were hybridized to Applied BiosystemsTM miRNA 3.1 Array Strip (20 h, 48 °C). Afterwards, the microarrays were rinsed and stained according to the technical protocol using the Affymetrix GeneAtlas Fluidics Station (Affymetrix, Santa Clara, CA, USA). The array strips were scanned using an Imaging Station of GeneAtlas System (Thermo Fisher Scientific, MA, USA).

Total RNA was isolated using the miRCURY RNA Isolation kit (Exiqon-Qiagen, United States). The total RNA concentration was determined by measuring the absorbance at 260 nm in a spectrophotometer (NanoDrop Technologies, United States). The rat microRNA content was evaluated using the Affymetrix GeneChip^TM miRNA Arrays 4.0 kit (Thermo Fisher Scientific, United States) containing probes for 728 mature and 490 rat pro-miRNAs.
RNA was biotin-labeled with a FlashTag^TM Biotin HSR RNA Labeling Kit (Thermo Fisher Scientific, United States). Labeled RNA samples were hybridized to the chip in a GeneChip^TM Hybridization Oven 645 (Thermo Fisher Scientific, United States) (18 h at 48°C and 60 rpm).
After hybridization, miRNA arrays were washed and stained using the Affymetrix GeneChip^TM Hybridization Wash and Stain Kit on GeneChip^TM Fluidics Station 450 and were then scanned with the Affymetrix GeneChip^TM Scanner 3000 7G (Thermo Fisher Scientific, United States), according to the protocol of the manufacturer. The data were processed using the Transcriptome Analysis Console software 4.0.1 (Thermo Fisher Scientific, United States).

The profiling of miRNA expression was performed using Affymetrix platform-based microarrays with GeneChip™ miRNA 4.1 Array Strip (Thermo Fisher Scientific, Waltham, MA, USA). The detailed technical procedure is described elsewhere [93 (link),94 (link)]. Each microarray was designed following the miRBase Release 20 database, including complementary probes for 2578 human mature miRNA, 1996 human snoRNA, CDBox RNA, H/ACA Box RNA, scaRNA, and 2025 human pre-miRNA. Preparation of miRNA to hybridization step was performed using the FlashTagTM Biotin HSR RNA Labeling Kit (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, 150 ng of miRNA was subjected to the poly(A) tailing and biotin ligation under the producer’s protocol. Biotin-labeled miRNA were then hybridized to GeneChip™ miRNA 4.1 Array Strip (20 h, 48 °C). Microarrays were then washed and stained according to the technical protocol using the Affymetrix GeneAtlas Fluidics Station (Affymetrix, Santa Clara, CA, USA). The array strips were scanned using an Imaging Station of GeneAtlas System (Thermo Fisher Scientific, Waltham, MA, USA).

Total RNA, including miRNA, was isolated from the spinal cord samples obtained from sALS patients and controls using a miRNeasy Mini Kit (Qiagen, Hilden, Germany). The quality of total RNA (RNA integrity number > 7.0) was assessed using a 2100 Bioanalyzer (Agilent Technology, Santa Clara, CA, USA). For each experimental group, 1 μg of pooled total RNA was labeled with biotin using a FlashTagTM Biotin HSR RNA Labeling Kit (Thermo Fisher Scientific, Waltham, MA, USA). The labeled RNA was then hybridized to a GeneChip^® miRNA 4.0 Array (Affymetrix, Santa Clara, CA, USA) in a GeneChip^® Hybridization Oven 645 (Affymetrix). The microarray was washed using GeneChip^® Fluidics Station 450 (Affymetrix) and scanned with a GeneChip^® Scanner 3000 7G (Affymetrix). The resulting data were analyzed using an Affymetrix^® GeneChip^® Command Console 4.0 (Affymetrix). Low signal-to-noise data were eliminated from the raw data. MiRNAs with changes in their levels greater than twofold were selected, and the changes were confirmed by real-time RT-PCR analysis.

The Affymetrix microRNA GeneChip Array plates (Affymetrix, Thermo Fisher) were used in this study according to the manufacturer’s instructions at the core facility for Bioinformatics and Expression Analysis (BEA, Karolinska Institutet, Stockholm, Sweden). In brief, two hundred micrograms of total RNA from bladder samples (collected after 6 h, 2 days and 8 days post wounding) were biotin-labeled for the Affymetrix GeneChip microRNA array 4.1 using the FlashTag biotin-HSR RNA labeling kit (Thermo Fisher Scientific). Each microarray contained sequences to interrogate all mature microRNA sequences in MiRBase Release 17. Hybridization, washing, and scanning of slides were performed according to the Affymetrix protocols. The scanned images were processed using the Affymetrix GeneChip Command Console. CEL files from scanning were imported to Transcriptome Analysis Console (TAC v4.00) and analyzed using the RMA method, that generated signals and Detection Above Background (DABG) p-values. Wounded and non-wounded bladders were compared for each time point using the Moderate t-test and the false discovery rates were calculated.

Total RNA of clone SS109 was extracted from hiPSC-CMs in different stages of maturation using TRIzol reagent followed by isolation and purification using miRNeasy (QIAGEN, USA) according to the manufacturer's instructions. RNA quality and quantity were assessed using Agilent 2100 Bioanalyzer chip (AGILENT, USA). All samples were labeled using FlashTag Biotin HSR RNA Labeling Kit (901,911, Thermo Fisher Scientific). To assess gene expression, RNA was hybridized to Clariom™ S Assay HT, human (902,969, Thermo Fisher Scientific), whereas to assess miRNA expression the biotinylated samples were hybridized to GeneChipTM miRNA 4.1 array plate (902,409, Thermo Fisher Scientific) washed and stained according to the manufacturer’s protocols. The gene and miRNA Array Plates were scanned using the GeneTitanTM Instrument (Thermo Fisher Scientific). Expression Console Software was used to create summarized expression values (CHP-files). Data were analyzed using Transcriptome Analysis Console (TAC) software generating fold-change, p-value, etc., and MATLAB homemade scripts (files are in NCBI Gene Expression Omnibus database repository record GSE188749).