RNA-Seq was performed on tissue isolated from 8–12 week old naïve male mice. RNA was extracted from the BLC with the miRVANA PARIS RNA kit (Life Technologies, Carlsbad, CA) and 100 ng from each sample (N=4) was used for library preparation with the TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA). Library preparation and 150 bp paired-end sequencing on the Illumina NextSeq 500 were performed by the Scripps Florida Genomics Core. Reads were mapped to the genome using TopHat/Bowtie software(34 (link), 35 (link)) and normalized means were obtained using the DeSeq package (36 (link)) in Bioconductor. RPKM values were used to determine basal levels of myosin II heavy chains.
Mirvana paris rna kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MirVana PARIS RNA kit is a product designed for the isolation and purification of total RNA, including small RNAs such as miRNA, from various sample types. It utilizes a proprietary methodology to extract and preserve RNA, maintaining its integrity for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
First strand cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Fast sybr green pcr kit, by Thermo Fisher Scientific (3 mentions)
Abi prism 7300 rt pcr system, by Thermo Fisher Scientific (3 mentions)
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Truseq rna sample prep kit, by Illumina (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Microrna reverse transcription kit, by EZBioscience (1 mentions)
5 protocols using mirvana paris rna kit
1
RNA-Seq Analysis of Murine BLC
2
Extraction and Quantification of Serum miRNA
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Total RNA was extracted using the mirVana PARIS RNA kit, according to the manufacturer instructions (Life Technologies, California, USA) using 500 μL of serum. After the denaturation, 25 fmol of synthetic miRNA cel-miR-23843 (link) (where “cel” corresponds to the abbreviation of species Caenorhabditis elegans) was added, which was used as quality control of RNA extraction and RT-qPCR.
3
Isolation and Quantification of miRNA and mRNA
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miRNA was isolated from EVs, tissues, and cells using the mirVanaTM PARISTM RNA kit (AM1556, Invitrogen). For mRNA analysis, First Strand cDNA Synthesis Kit (K1622, Fermentas Inc., Hanover, MD) was used to randomly synthesize cDNA from 1 μg of total RNA. For miRNA analysis, TaqMan microRNA Reverse Transcription Kit (4366597, Applied Biosystems Inc. Carlsbad, CA) was used to synthesize cDNA of miRNA. Next, RNA was quantitatively analyzed using Fast SYBR Green PCR kit (Applied Biosystems) and ABI PRISM 7300 RT-PCR system (Applied Biosystems), with three repeated wells for each sample. U6 served as the internal reference for miR-138-5p, while GAPDH for the remaining genes. All primers were purchased from Sangon Biotechnology (Shanghai, China), and the sequences are shown in Supplementary Table 1 . Besides, reverse primers of TaqManTM microRNA Reverse Transcription Kit were used.
4
Comprehensive miRNA and mRNA Analysis
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According to the manufacturer's instructions, miRNA was isolated from EVs, tissues, and cells by using the mirVanaTM PARISTM RNA kit (AM1556, Invitrogen, CA, USA). For mRNA analysis, cDNA was synthesized randomly from 1 μg of total RNA using the First Strand cDNA Synthesis Kit (K1622, Fermentas, USA) was used to synthesize cDNA randomly from 1 μg of total RNA; for miRNA analysis, the TaqManTM MicroRNA Reverse Transcription Kit (4366597, Applied Biosystems, USA) was used to synthesize cDNA for miRNA. Finally, the Fast SYBR Green PCR kit (Applied biosystems) and the ABI PRISM 7300 RT-PCR system (Applied biosystems) were used to analyze RNA quantitatively, and each sample was repeated for three replicate wells. miR-30c was used with U6 as the internal reference, and the remaining genes were used with GAPDH. The relative gene expression was analyzed by the 2-ΔΔCt method, calculated as △Ct=CT (target gene)−CT (internal reference) and △△Ct=△Ct (Experimental group)−△Ct (control group), repeated the experiment 3 times, and took the average value. All primers were purchased from Sangon Biotech (Shanghai) Co., Ltd. and the primer sequences were as follows Table 1 .
5
Quantifying miRNA Expression via qRT-PCR
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Based on the protocols of the mirVanaTM PARISTM RNA kit (AM1556, Invitrogen), miRNA was isolated from exosomes, tissues, and cells. Total RNA (1 µg) was reversely transcribed into cDNA utilizing the First Strand cDNA Synthesis Kit (K1622, Fermentas) for mRNA analysis, while the cDNA for miRNA analysis was synthesized with a polyA-tailing MicroRNA Reverse Transcription Kit (EZB-miRT2-S, EZBioscience, Roseville, MN). After that, the Fast SYBR Green PCR kit (Applied Biosystems) and ABI PRISM 7300 RT-PCR system (Applied biosystems) were employed for the qRT-PCR assay, and three replicate wells set for each sample. U6 was used as a housekeeper gene for miR-30c, and GAPDH was for other genes. Further, the 2−ΔΔCt method was used to calculate the relative transcription level of target genes. Involved primers were commercially obtained (Sangon Biotech, Shanghai, China), as listed in Supplementary Table 1 .
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