Ascites cells were collected and total cellular RNA was purified using the mirVana RNA Purification Kit following manufacturer’s (Thermo Fisher) recommendations. RNA yield and purity were determined on an Agilent Model 2100 Bioanalyzer. Sufficient yield was obtained with an RNA Integrity Number (RIN) >9.0 to proceed with RNA sequencing.
Mirvana rna purification kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MirVana RNA purification kit is a product designed for the isolation and purification of RNA from various biological samples. It utilizes a guanidinium-based lysis and phenol-chloroform extraction method to extract and purify high-quality RNA, including small RNAs such as microRNAs. The kit provides reagents and protocols for the efficient isolation of RNA from cells, tissues, and other sample types.
Automatically generated - may contain errors
Lab products found in correlation
Agilent model 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Dropsense 16 spectrophotometer, by Agilent Technologies (1 mentions)
Hiseq 4000 genome sequencing platform, by Illumina (1 mentions)
Morpholino oligonucleotide, by Gene Tools (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
5 protocols using mirvana rna purification kit
1
RNA Isolation from Ascites Cells
2
Tumor-Derived RNA Purification and Analysis
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Whole cell RNA was purified from the appropriate tumors using the mirVana RNA purification kit according to manufacturer’s instructions (Thermo Fisher). Yield and purity were assessed in the University of Iowa Institute of Human Genetics (IIHG) using a Trinean DropSense 16 and an Agilent Model 2100 Bioanalyzer.
3
Total RNA Extraction and RNA-Seq Library Preparation
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Total RNA was extracted from normal control and tumor tissues using the mirVana RNA purification kit (Thermo Fisher, Waltham, MA, USA) following manufacturer’s instructions. We initially evaluated RNA quality with NanoDrop Model 2000 spectrophotometer and used a 260 nm/280 nm absorbance ratio of ~2.0. RNAs with an RNA integrity number (RIN)26 (link) of ≥7.0 via Trinean DropSense 16 spectrophotometer and Agilent Model 2100 bioanalyzer were then used for RNA sequencing. Messenger RNA-focused sequencing libraries were constructed using 500 ng of total RNA per sample that was converted to cDNA and linked to specific adaptors for sequencing via the Illumina TruSeq RNA library prep kit (Illiumina, San Diego, CA, USA). Indexed libraries for sequencing were re-concentrated into equimolar aliquots using the Agilent Model 2100 bioanalyzer and validated via an Illumina Library Quantification Kit (KAPA Biosystems, Wilmington, MA, USA). The Illumina HiSeq. 4000 genome sequencing platform employing 150 bp paired-end sequencing by synthesis chemistry was used. All library preparation and sequencing procedures were performed in the Genome Core Facility of the University of Iowa Institute of Human Genetics27 (link).
4
Morpholino-Mediated Knockdown and Rescue in Zebrafish
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Morpholino oligonucleotides (Gene Tools) were re-suspended in water to a stock concentration of 0.5 mM. Final morpholino concentrations were injected into 1–2 cell stage embryos in 3 nl amounts and embryos were maintained at 28.5°C. The DUOX morpholino was used as published (Yoo et al., 2012 (link)). The vimentin and Larp6 splice blocking morpholinos used were as follows:
vim MO1 targeting Exon 1- Intron 1 (80 µm):
5’-GTAATAGTGCCAGAACAGACCTTCTC-3’
vim MO2 targeting Intron 1-Exon 2 (110 µm):
5’-TCTTGAAGTCTGGAAATGAGATGCA-3’
larp6 MO targeting Exon 2-Intron 2 (80 µm):
5’-GGGTGTTGGTCTTACCTTCTTGAA-3’ control MO
5’-CCTCTTACCTCAGTTACAATTTATA-3’
To analyze knockdowns, RT-PCR was performed on RNA isolated from single larvae at 2 dpf. The following primers were used to assess knockdown:
Vim MO 1 and 2:
F-5’-ACCGGGGAAAAGAGCAAAGT-3’ R-5’-CGAGCCAGAGAGGCGTTATC-3’
Larp6 MO:
F-5’-CAAACTGGGCTTCGTCAGTG-3’ R-5’-TCCGTTGTTGGAATCTCCGC-3’
Rescue experiments were performed using zebrafish vim RNA. In short, a gene block (IDT) for vimentin was used as the template. The sequence was cloned into the PCS2 +vector from which in vitro transcription was performed (Ambion). RNA was purified with the miRvana RNA purification kit (Ambion). RNA was injected at 125 ng/µl at the 1–2 cell stage in 3 nl amounts.
5’-GTAATAGTGCCAGAACAGACCTTCTC-3’
5’-TCTTGAAGTCTGGAAATGAGATGCA-3’
5’-GGGTGTTGGTCTTACCTTCTTGAA-3’ control MO
5’-CCTCTTACCTCAGTTACAATTTATA-3’
To analyze knockdowns, RT-PCR was performed on RNA isolated from single larvae at 2 dpf. The following primers were used to assess knockdown:
Vim MO 1 and 2:
F-5’-ACCGGGGAAAAGAGCAAAGT-3’ R-5’-CGAGCCAGAGAGGCGTTATC-3’
Larp6 MO:
F-5’-CAAACTGGGCTTCGTCAGTG-3’ R-5’-TCCGTTGTTGGAATCTCCGC-3’
Rescue experiments were performed using zebrafish vim RNA. In short, a gene block (IDT) for vimentin was used as the template. The sequence was cloned into the PCS2 +vector from which in vitro transcription was performed (Ambion). RNA was purified with the miRvana RNA purification kit (Ambion). RNA was injected at 125 ng/µl at the 1–2 cell stage in 3 nl amounts.
5
Fin Regeneration Gene Expression
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RNA was extracted from an approximately 500 µm length portion of the fin using the miRvana RNA purification kit (Ambion). First strand cDNA synthesis was performed using super script III (Invitrogen). Resulting cDNA was diluted 1:10 in RNAse free water before qRT-PCR was performed in a minimum of triplicate using Roche green master mix (Roche) for rps11, col1a1 and col2a2 from purified fin RNA. Fold change was determined using efficiency-corrected comparative quantitation. Data were normalized to no wound age matched control samples. Primers:
Rps11:
F-5’-TAAGAAATGCCCCTTCACTG-3’ R-5’-GTCTCTTCTCAAAACGGTTG-3’
Col1a1:
F-5’-TGTCACTGAGGATGGTTGCAC-3’ R-5’-GCAGACGGGATGTTTTCGTTG-3’
Col2a1b (Durán et al., 2011 (link)):
F- 5’-AACAGAAGTGCTTCCGAACG-3’ R- 5’-TGCTCTGGTTTCTCCCTCAT-3’
Rps11:
F-5’-TAAGAAATGCCCCTTCACTG-3’ R-5’-GTCTCTTCTCAAAACGGTTG-3’
Col1a1:
F-5’-TGTCACTGAGGATGGTTGCAC-3’ R-5’-GCAGACGGGATGTTTTCGTTG-3’
Col2a1b (Durán et al., 2011 (link)):
F- 5’-AACAGAAGTGCTTCCGAACG-3’ R- 5’-TGCTCTGGTTTCTCCCTCAT-3’
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