Total RNA was isolated from the laser-microdissected tissue fragments using the RNeasy FFPE kit (Qiagen), and RNA quality and quantity were determined with the 2100 Bioanalyzer and RNA 6000 Pico kit (Agilent Technologies) following the instructions of the manufacturers. Total RNA (40 ng) from each tissue fraction was amplified with the Ovation PicoSL WTA System V2 in combination with the Encore Biotin Module (Tecan). Amplified cDNAs were hybridized on Mouse Gene 1.0 ST arrays (Affymetrix), and staining and scanning were done according to the Affymetrix expression protocol including minor modifications as suggested in the Encore Biotin protocol. Expression console (Affymetrix) was used for quality control and to obtain annotated normalized RMA gene-level data (standard settings including sketch-quantile normalization). Fluorescence-activated cell sorting (FACS) and microarray analysis of E13.5/E14.5 Pitx3GFP/+ mdDA neurons were reported by Fukusumi et al. (2015) (link).
Ovation picosl wta system v2
Manufactured by Tecan
Sourced in United States
The Ovation PicoSL WTA System V2 is a laboratory equipment designed for whole transcriptome amplification from low-input RNA samples. It is a specialized tool for generating amplified cDNA from small amounts of RNA, enabling downstream applications such as gene expression analysis and next-generation sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Encore biotin module, by Tecan (10 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (8 mentions)
Rneasy micro kit, by Qiagen (7 mentions)
Encore biotinil module, by Tecan (5 mentions)
Qiashredder, by Qiagen (4 mentions)
Rna nano, by Agilent Technologies (4 mentions)
Superscript 3, by Thermo Fisher Scientific (4 mentions)
Rneasy, by Qiagen (4 mentions)
Nanodrop spectrophotometer, by Agilent Technologies (4 mentions)
Gotaq qpcr master mix, by Promega (4 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Expression console, by Thermo Fisher Scientific (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Genechip scanner 3000 7g, by Thermo Fisher Scientific (3 mentions)
Qiaquick pcr purification kit, by Qiagen (3 mentions)
Genomestudio, by Illumina (3 mentions)
Iq sybr green supermix, by Bio-Rad (3 mentions)
Agilent rna 6000 pico kit, by Agilent Technologies (3 mentions)
Rneasy plus micro kit, by Qiagen (3 mentions)
53 protocols using ovation picosl wta system v2
1
Microdissected Tissue RNA Profiling
2
Laser Microdissection of Uterine Epithelia
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Laser microdissection was performed as previously described.4 , 19 To isolate the luminal epithelium, frozen sections of pregnant uteri (20 µm) were mounted on film slides and dissected using the LMD7000 System (Leica Microsystems, Wetzlar, Germany).4 , 19 For each day of pregnancy, tissues from 3 females were dissected. The extracted RNA was amplified using the Ovation PicoSL WTA System V2 (Tecan, Zürich, Switzerland).
3
Optimized RNA Extraction and qPCR Analysis
4
Transcriptional Changes in Isolated Th17 and Treg Cells upon CD95L Treatment
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Isolated Th17 and Treg cells from two healthy donors were treated with or without 100 ng/mL of cl-CD95L for 8 hr. Total RNA was extracted with the Nucleospin RNA XS kit (Macherey-Nagel), and quality was assessed with the RNA6000 nano chip (Agilent). For each condition, 9 ng of RNA was reverse transcribed with the Ovation PicoSL WTA System V2 (Nugen, Leek, The Netherlands). Fragmented cDNAs were hybridized to GeneChip Human Gene 2.0 ST microarrays (Affymetrix). Then chips were scanned on a GeneChip Scanner 3000 7G (Affymetrix). Raw data and quality-control metrics were generated from scanned images using the Expression Console software (Affymetrix).
Probes were mapped with Brainarray V20 CDF files (http://brainarray.mbni.med.umich.edu/ ) and normalized by robust multi-array averaging with R software. Statistical analyses were performed with Partek Genomics Suite; a p value ≤ 0,05 was considered significant. Pathway enrichment analyses were generated with Ingenuity Pathway Analysis (QIAGEN).
Probes were mapped with Brainarray V20 CDF files (
5
Laser Microdissection for RNA Isolation
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Root sections were collected on ultraviolet-sterilized and RNase-free polyester-membrane 0.9 µm FrameSlides (Leica). ECs and plaques were collected using a laser microdissection system (LMD7000, Leica) in RNase-free tubes. RNA was isolated with the PAXgene RNA MinElute kit (Qiagen) and followed by pre-amplification and reverse transcription with Ovation PicoSL WTA System V2 (NuGEN) following manufacturer’s instructions.
6
Illumina Expression Profiling of Mouse Transcriptome
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Total RNA (about 10 ng) was amplified using the Ovation PicoSL WTA System V2 in combination with the Encore Biotin IL Module (Nugen, San Carlos, CA, USA). In all, 1000 ng of amplified cDNA was hybridized to Mouse Ref-8 v2.0 Expression Bead Chips (Illumina, San Diego, CA, USA). Staining and scanning were done according to the Illumina expression protocol. Data were processed using the GenomeStudioV2011.1 software (San Diego, CA, USA; gene expression module version 1.9.0) in combination with the Mouse Ref-8_V2_0_R3_11278551_A.bgx annotation file. The background subtraction option was used and an offset to remove remaining negative expression values was introduced. CARMAweb was used for quantile normalization.64 (link)
7
Quantification of Muscle Gene Expression
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Total RNA was extracted from Gastrocnemius muscle using a miRCURY RNA isolation kit (Exiqon). Muscle RNA was reversely transcribed and amplified using Ovation PicoSL WTA System V2 (Nugen). Quantitative PCR was performed using a commercial SYBR green mastermix (Biorad) and specific primers for VEGF-B as described by Hagberg et al. [23 (link)] and for VEGFR1, IRS1 and Glut4. Primers for IRS1 were described in Meijer et al. [29 (link)], primers for VEGFR1 were described in Robciuc et al. [32 (link)]. Data were corrected for the geometric mean of Rps15 and expressed relative to the muscle of vehicle-treated, chow-fed mice.
8
Quantitative Gene Expression in Melanocytes and Keratinocytes
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In a tissue section, ~200 irradiated melanocytes or keratinocytes were captured by LCM. Total RNA was isolated from each of these two types of cells using a single cell RNA and RNase-free DNase kit (Norgen Biotek Corp.). Total RNA (5 μl) was then subject to amplification using the Ovation PicoSL WTA System V2 (NuGEN Technologies, Inc.), and the cDNA purified by using QIAGENs’ MinElute Reaction Cleanup Kit. Concentration of the purified cDNA was measured by using the NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Inc.).
The TaqMan Gene Expression Assay system (Life Technologies) was used for quantitating transcriptional levels of the following genes: MITF (Hs01117294_m1), MLANA (MART1) (Hs00194133_m1), KRT10 (Hs01043114_g1), and KRT14 (Hs00265033_m1). The 18s rRNA gene (Hs03003631_g1) was used as an endogenous control, and, an RNA sample for the expression of a selected gene was run in triplicate along with a no-template-control. PCR reactions were performed on BIO-RADs’ CFX96 Real-Time System in 20 μl volumes at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Relative quantitation of mRNA expression was calculated by the 2-ΔCt method [17 (link)]
The TaqMan Gene Expression Assay system (Life Technologies) was used for quantitating transcriptional levels of the following genes: MITF (Hs01117294_m1), MLANA (MART1) (Hs00194133_m1), KRT10 (Hs01043114_g1), and KRT14 (Hs00265033_m1). The 18s rRNA gene (Hs03003631_g1) was used as an endogenous control, and, an RNA sample for the expression of a selected gene was run in triplicate along with a no-template-control. PCR reactions were performed on BIO-RADs’ CFX96 Real-Time System in 20 μl volumes at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Relative quantitation of mRNA expression was calculated by the 2-ΔCt method [17 (link)]
9
Quantitative Analysis of GABA Receptor Expression
10
Gene Expression Profiling by Microarray
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For gene profiling, total RNA was extracted using miRNeasy Mini kit (Qiagen, #217004), RNA integrity was checked using Agilent 2100 Bioanalyzer (Agilent RNA 6000 Pico Kit), and cDNA was amplified with the Ovation PicoSL WTA System V2 (Nugen, 3312) in combination with the Encore Biotin Module (Nugen, USA). Amplified cDNA was hybridized on GeneChip™ Human Gene 2.0 ST arrays (Affymetrix, 902113). Expression console (v.1.3.0.187, Affymetrix) was used for quality control. All subsequent computational analysis was performed in R using Bioconductor packages. Expression data were RMA normalized using the oligo package (version 1.38.0) and probe sets were annotated using the package hugene20sttranscriptcluster.db (version 8.5.0). Differential expression analyses were performed using the limma package (version 3.30.7) and P-values were adjusted for multiple testing by Benjamini-Hochberg correction. A gene was considered as differentially expressed if the adjusted p-value (FDR) was below a threshold of 0.05 and the fold-change was greater than or equal to 2. Functional enrichments were conducted using HOMER [55] (link). Gene set enrichment analysis was performed using GSEA 3.0 [56] , [57] (link) with genes ranked by log2 ratio between XM001 PPs and XM001 iPSCs.
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