RNA was isolated from the vaginal cells from two treated and two control macaques using RNeasy Micro spin columns (Qiagen) and measured for quality using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). All samples had a RIN score between 7 and 9.6 (average: 8.7). Sequencing libraries were prepared with TruSeq RNA v2 kits (Illumina, San Diego, CA) using the manufacturer’s standard protocols. Library quality was assessed by measuring insert size using a Bioanalyzer 2100 (average size: 323.6bp) and library concentration determined by qPCR (Library Quantification Kit, KR0405, Kapa Biosystems, Wilmington, MA). The libraries were run on a HiSeq 2500 with 50bp paired end reads (average reads per sample: 13.68 million). Reads were trimmed of Illumina adapter sequences using Trimmomatic v0.32 46 (link). Trimmed paired end reads were mapped against the macaque genome (MMUL 1.0, v78) using the STAR aligner v2.3.0e 47 (link). Transcript abundances were estimated using HTSeq v0.6.1 48 (link). The count matrices were then tested for differentially expressed genes using edgeR v3.0.2 49 (link) and Student’s t test. Given the small dataset, the genes with p value and false discovery rate (FDR) < 0.05 were then tested for enriched pathways using Fisher’s exact test (p < 0.05 significance level).
Rneasy micro spin column
Manufactured by Qiagen
Sourced in United Kingdom
The RNeasy Micro spin columns are a laboratory tool designed for the purification of total RNA from a small number of cells or a small amount of tissue. The columns utilize a silica-based membrane to efficiently capture and purify RNA molecules, enabling the user to obtain high-quality RNA samples for various downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Bioanalyzer 2100, by Roche (2 mentions)
Library quantification kit, by Roche (2 mentions)
Truseq rna v2 kit, by Illumina (2 mentions)
Rneasy micro kit, by Qiagen (2 mentions)
Palm adhesivecap, by Zeiss (2 mentions)
Agilent rna 6000 pico labchip, by Agilent Technologies (2 mentions)
Rlt plus buffer, by Qiagen (2 mentions)
Qubit rna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Avantor (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Rna 6000 pico labchip reagent set, by Agilent Technologies (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Trizol, by Qiagen (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
8 protocols using rneasy micro spin column
1
Macaque Vaginal Transcriptome Analysis
2
RNA Extraction and cDNA Synthesis
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RNA was isolated from cell cultures using the miRNeasy mini-kit (Qiagen, Hilden, Germany) according to the protocol described by the manufacturer. To obtain a higher yield of RNA, we used RNeasy micro-spin columns (Qiagen). RNA concentration was determined with the NanoDrop (Peqlab Biotechnologie GmbH, Erlangen, Germany). Isolated RNA was stored at −80 °C.
Trancription of RNA into complementary DNA (cDNA) was performed with the High-Capacity cDNA Reverse Transcription Kit (Life Technologies GmbH) according to the manufacturer‘s instructions. For each reaction (20 µL) 200 ng RNA were used.
Trancription of RNA into complementary DNA (cDNA) was performed with the High-Capacity cDNA Reverse Transcription Kit (Life Technologies GmbH) according to the manufacturer‘s instructions. For each reaction (20 µL) 200 ng RNA were used.
3
Porcine Lung RNA Extraction Protocol
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Approximately 200 mg of porcine lung tissue was homogenized in 800 µL RLT buffer (Qiagen, Hilden, Germany) using Precellys CK14 ceramic beads (2 cycles of 20 s at 6500 r.p.m.; 15 s break). Next, 1.4 mL of Trizol reagent was added to 150 µL of homogenate. After 5 min at room temperature, 280 µL of chloroform was added and the samples were again incubated at room temperature for 2 min. Phase separation was achieved by 15 min centrifugation at 12,000 × g at 4 °C. An equal amount of 70% ethanol was added to the aqueous supernatant and the mixture was applied to RNeasy Microspin columns (RNeasy Micro Kit, QIAGEN, Hilden, Germany), followed by an on-column DNase digestion and several wash steps. Finally, total RNA was eluted in 14 μL of nuclease-free water. Purity and integrity of the RNA was assessed on the Agilent 2100 Bioanalyzer with the RNA 6000 Pico LabChip reagent set (Agilent, Palo Alto, CA, USA).
4
Laser Capture Microdissection and RNA Extraction
5
RNA Extraction from Laser-Captured Cells
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Cells were laser captured onto Zeiss PALM AdhesiveCap (Carl Zeiss GmbH, Germany), and RNA was extracted using the RNEasy micro kit (Qiagen, Crawley, United Kingdom). The AdhesiveCap tubes were closed, inverted, and incubated for 30 minutes at room temperature. The lysates were then centrifuged at 12 000 rpm for 5 minutes. Samples were stored at −80°C for batch RNA extraction and purification. Total RNA was prepared using RNEasy micro spin columns (Qiagen, Crawley, United Kingdom), with on-column DNase I digestion to remove genomic DNA contamination. RNA concentrations and qualities were assessed using the Agilent RNA 6000 Pico LabChip on the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). Samples with RNA integrity number (RIN) <5 were considered of insufficient quality and excluded from downstream RNA amplification, biotinylation, and gene expression microarray analysis.
6
Macaque Vaginal Transcriptome Analysis
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RNA was isolated from the vaginal cells from two treated and two control macaques using RNeasy Micro spin columns (Qiagen) and measured for quality using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). All samples had a RIN score between 7 and 9.6 (average: 8.7). Sequencing libraries were prepared with TruSeq RNA v2 kits (Illumina, San Diego, CA) using the manufacturer’s standard protocols. Library quality was assessed by measuring insert size using a Bioanalyzer 2100 (average size: 323.6bp) and library concentration determined by qPCR (Library Quantification Kit, KR0405, Kapa Biosystems, Wilmington, MA). The libraries were run on a HiSeq 2500 with 50bp paired end reads (average reads per sample: 13.68 million). Reads were trimmed of Illumina adapter sequences using Trimmomatic v0.32 46 (link). Trimmed paired end reads were mapped against the macaque genome (MMUL 1.0, v78) using the STAR aligner v2.3.0e 47 (link). Transcript abundances were estimated using HTSeq v0.6.1 48 (link). The count matrices were then tested for differentially expressed genes using edgeR v3.0.2 49 (link) and Student’s t test. Given the small dataset, the genes with p value and false discovery rate (FDR) < 0.05 were then tested for enriched pathways using Fisher’s exact test (p < 0.05 significance level).
7
Total RNA Extraction from Gonad Tissue
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For bulk gonad tissue (both freshly isolated and ex vivo cultured), gonads were first homogenized in a 1.5 mL centrifuge tube in 100 μL TRIzol (Invitrogen 15596026) using a motorized pestle. After bringing the final volume to 1 mL with TRIzol, samples were vortexed on high for 30 s before storing at –80 °C. To extract total RNA from TRIzol samples, 200 μL of chloroform was added per sample. Samples were vortexed vigorously for 30 s and then centrifuged at 12,000 x g for 15 min at 4 °C. The upper aqueous layer was carefully extracted, mixed 1:1 with an equal volume of fresh 70% ethanol, and loaded onto a QIAgen RNeasy Micro spin column for further cleanup and elution. The QIAgen RNeasy Micro Kit (cat. no. 74004) protocol was followed exactly, including the on-column DNase digestion for 15 min. For FACS-sorted cell samples that were sorted into QIAgen RLT Plus Buffer, samples were mixed with equal volume of 70% ethanol and loaded onto column without TRIzol homogenization and chloroform extraction. RNA concentrations were quantified using the Qubit RNA HS Assay Kit (ThermoFisher cat. no. Q32852) following the manufacturer’s protocol.
8
RNA Extraction from Gonad Tissue
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For bulk gonad tissue (both freshly isolated and ex vivo cultured), gonads were first homogenized in a 1.5 mL centrifuge tube in 100 μL TRIzol (Invitrogen 15596026) using a motorized pestle. After bringing the final volume to 1 mL with TRIzol, samples were vortexed on high for 30 seconds before storing at −80°C. To extract total RNA from TRIzol samples, 200 μL of chloroform was added per sample. Samples were vortexed vigorously for 30 seconds and then centrifuged at 12,000xg for 15 min at 4°C. The upper aqueous layer was carefully extracted, mixed 1:1 with an equal volume of fresh 70% ethanol, and loaded onto a QIAgen RNeasy Micro spin column for further cleanup and elution. The QIAgen RNeasy Micro Kit (cat. no. 74004) protocol was followed exactly, including the on-column DNase digestion for 15 min. For FACS-sorted cell samples that were sorted into QIAgen RLT Plus Buffer, samples were mixed with equal volume of 70% ethanol and loaded onto column without TRIzol homogenization and chloroform extraction. RNA concentrations were quantified using the Qubit RNA HS Assay Kit (ThermoFisher cat. no. Q32852) following the manufacturer’s protocol.
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