Total RNA was isolated from cultured cells using TRIzol (TaKaRa, Tokyo, JPN) and purified using miRNeasy columns (Qiagen). All RNA isolation and quantitative real-time PCR were performed according to our previous studies1 (link),2 (link),20 (link). See Supplementary Material for details.
Mirneasy column
Manufactured by Qiagen
Sourced in United Kingdom
The MiRNeasy columns are a core component of Qiagen's RNA extraction and purification solutions. They are designed to efficiently isolate and purify microRNA (miRNA) from a variety of sample types. The columns utilize a specialized silica-based membrane to selectively bind and capture miRNA molecules, enabling their separation from other RNA species present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Qiazol, by Qiagen (7 mentions)
Mirneasy mini kit, by Qiagen (7 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (3 mentions)
Experion, by Bio-Rad (2 mentions)
Trizol reagent kit, by Thermo Fisher Scientific (2 mentions)
Hiseq 2500 sequencer, by Illumina (2 mentions)
Polytron pt1200e, by Kinematica (2 mentions)
Agilent bioanalyzer, by Agilent Technologies (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Trizol, by Takara Bio (1 mentions)
Power sybr green mix, by Thermo Fisher Scientific (1 mentions)
Perfecta sybr green supermix, by Quanta Biosciences (1 mentions)
Geneamp rna pcr kit, by Thermo Fisher Scientific (1 mentions)
Qscript mirna cdna synthesis kit, by Quanta Biosciences (1 mentions)
Viia 7 system, by Thermo Fisher Scientific (1 mentions)
Taqman pcr master mix, by Thermo Fisher Scientific (1 mentions)
37 protocols using mirneasy column
1
Total RNA Isolation and qRT-PCR
2
Real-Time qRT-PCR Analysis of miRNA and mRNA
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Real-time qRT-PCR analysis was performed as previously described11 (link)45 (link). Briefly, RNA was extracted using miRNeasy columns (Qiagen, Valencia, CA). miRNA expression analysis was performed using the qScript miRNA cDNA synthesis kit (Quanta Biosciences, Gaithersburg, MD) and PerfeCTa SYBR Green Supermix (Quanta Biosciences). miRNAs were amplified using specific mature miRNA sequences as forward primers and the universal primer provided in the kit as the reverse primer. U6 was used as internal control. A GeneAmp RNA PCR kit (Applied Biosystems, Carlsbad, CA) and POWER SYBR Green mix (Applied Biosystems) were used for mRNA quantification. PCR primer sequences are in Supplementary Table 3 .
3
Quantitative Analysis of Exosomal RNA
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RNA from EXOs was isolated using miRNeasy columns (Qiagen) according to the manufacturer’s protocol; total RNA from cell lines was extracted with a TRIzol extraction kit (Life Technologies, Grand Island, NY, USA). Nucleic acid quality and quantity were assessed with a NanoDrop spectrophotometer (NanoDrop Technologies, ThermoFisher Scientific). Total RNA for each sample was reverse transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit (Life Technologies) according to the manufacturer’s protocols. qPCR was performed on a ViiA7 system (Life Technologies) using TaqMan PCR Master Mix (Life Technologies). Predesigned TaqMan probes (Life Technologies) were used for Matrix Metalloproteinase 1 (MMP1, Hs00899658_m1), Matrix Metalloproteinase 9 (MMP9, Hs00957562_m1), Cyclin D1 (CCND1, Hs00765533_m1), CD99 (Hs00908458) and miR-199a-3p (Hs002304). Relative quantification as performed with the ΔΔCT method, and the expression levels of the target genes were normalized to those of the housekeeping gene GAPDH (Hs99999905_m1), RNU6b (Hs001093) or miR-16 (Hs000391).
4
RNA and DNA Extraction Protocol
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Total RNA was extracted from tissues using Qiazol, followed by purification on miRNeasy columns (Qiagen) according to the manufacturer’s recommendations. Genomic DNA was extracted from the remaining mix of phenol-chloroform using homemade protocol described in Additional file 2 .
Laser capture microdissection, qRT-PCR and other experimental procedures are described in Additional file2 .
Laser capture microdissection, qRT-PCR and other experimental procedures are described in Additional file
5
Total RNA Extraction from Thyroid Tissue
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Total RNA was extracted from thyroid tissues using a Trizol reagent kit (Invitrogen), followed by purification on miRNeasy columns (Qiagen). The RNA concentration was spectrophotometrically quantified, and its integrity was verified by visualization using an automated electrophoresis system (Experion, Bio-Rad).
6
Yeast RNA Extraction and 4tU Labeling
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Yeast RNA was prepared by extraction with hot acidic phenol (19 ). Total RNA was extracted, and 4tU-containing mRNA was biotinylated and isolated as previously described (20 (link),21 (link)). To obtain high quality RNA, samples were further subjected to the clean-up protocol with the RNeasy Mini Kit (Qiagene, Cat. #74104), and quality was checked with gel electrophoresis. Nascent RNA was then biotinylated as described (20 (link),21 (link)), using 400 μl (∼40 μg) total RNA and 4 μg MTSEA biotin-XX (Biotium) and purified by MyOne Streptavidin C1 Dynabeads (Invitrogen). The isolated RNA was further purified and concentrated using Qiagen miRNeasy columns (#217084), according to the manufacturer's protocol. The isolated RNA was then prepared for sequencing using the Ovation SoLo or Ovation Universal RNA-seq System kits (Tecan), according to the manufacturer's instructions. Libraries were sequenced on the Illumina NextSeq high output platform using single reads 75 in Institute of Molecular Biology of Academia Sinica
7
miRNA Extraction and Sequencing
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miRNA was prepared from biological duplicates using TRIzol reagent (Life Technologies, cat.# 15596–026) following the manufacturer’s protocol, and then cleaned up on miRNeasy columns (Qiagen, cat.# 217004) with on-column DNA digestion. Sample integrity, preparation, and sequencing was performed at the Yale Center for Genome Analysis (West Haven, CT) using the Illumina Hi-Seq 2000 sequencing system.
8
RNA Isolation from Gonadal Adipose
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Flash-frozen gonadal adipose samples upon sacrifice were weighed and homogenized in QIAzol (Qiagen, Germantown, MD), and following chloroform phase separation, RNA was isolated according to the manufacturer's protocol using miRNeasy columns (Qiagen, Germantown, MD).
9
Analysis of Splice Variant Detection via RT-PCR
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Cells were transfected with the minicassette plasmid SXN13 (23 (link)) with Effectene (Qiagen). After 48 h, total RNA was extracted with miRNeasy columns (Qiagen) and cDNA prepared using oligo-dT and Superscript III (Life Technologies). PCR reactions to detect the splicing products were done using the primers SNXF: 5′-GACCATTCACCACATTGGTG-3′; and SXNRv: 5′-GAACCTCTGGGTCCAAGG-3′. PCR for specifically detecting partial splice products used SXNF and SXNJrv: 5′-GACCACCAGCAGCCTGGA-3′ or SKNJF: 5′-GCCCTGGGCAGGTCGAC-3′ and SXNRv. All products were electrophoresed in a 2% agarose gel.
PCR Primers (Table1 ):
PCR Primers (Table
10
Total RNA Extraction and Purification
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Total RNA was extracted using a TRIzol Reagent kit (Invitrogen) followed by purification on miRNeasy columns (Qiagen, Hilden, Germany). The RNA concentrations were spectrophotometrically quantified, and their integrity was verified using an automated electrophoresis system (Experion, Bio-Rad).
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