For high-throughput screening of miRNAs differentially expressed in aged mice, NGS was performed with total RNA isolated from the whole blood of mice. Total RNA was isolated from whole blood using the Hybride-R RNA Kit (GeneAll), according to the manufacturer’s instructions. Total RNA integrity was verified using an Agilent 2100 Bioanalyzer (Agilent Technologies), with an RNA integrity number (RIN) value >8 as a cutoff value. Small RNA sequencing libraries were prepared using a TruSeq Small RNA Library Preparation kit (Illumina), according to the manufacturer’s instructions. A small RNA (1 μg) sample pooled with equal amount of each mouse (n = 3) was ligated with RNA 3′ and RNA 5′ adapters. Reverse transcription followed by PCR was used to create cDNA constructs based on small RNA ligated to 3′ and 5′ adapters. This process selectively enriched fragments with adapter molecules on both ends. The small RNA fraction was purified with the Pippin Prep electrophoresis platform (Sage Science). The quality of the libraries was verified by capillary electrophoresis using an Agilent 2100 Bioanalyzer (Agilent Technologies). The libraries were pooled in equimolar amounts and loaded on the flow cell of a HiSeq 2000 sequencing system (Illumina). Sequencing was performed to generate 1 × 50-bp length read.
Hybride r rna kit
Manufactured by GeneAll
The Hybride-R RNA Kit is a laboratory product designed for the purification of high-quality RNA from a variety of sample types. It utilizes a hybrid method that combines both silica-based membrane and magnetic bead-based technologies. The kit provides efficient and reliable RNA extraction, suitable for downstream applications such as real-time PCR, RT-PCR, and other RNA-based analyses.
Automatically generated - may contain errors
Lab products found in correlation
Abi 7900ht real time pcr system, by Thermo Fisher Scientific (3 mentions)
Primescript 1st strand cdna synthesis kit, by Qiagen (2 mentions)
Sybr green pcr kit, by Qiagen (2 mentions)
Universal cdna synthesis kit 2, by Qiagen (2 mentions)
Truseq small rna library preparation kit, by Illumina (1 mentions)
Hiseq 2000 sequencing system, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
4 protocols using hybride r rna kit
1
High-throughput miRNA Profiling in Aged Mice
2
Quantitative mRNA Expression Analysis
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Total RNA from BEAS-2B and A549 cells was extracted using the Hybride-R™ RNA Kit (GeneAll®™, Seoul, Korea), as per the manufacturer’s instructions. For mRNA analysis, 1 μg of total RNA was reverse transcribed into cDNA using PrimeScript 1st strand cDNA Synthesis Kit (Exiqon, Vedbaek, Denmark). For the determination of mRNA expression, real-time polymerase chain reaction (RT-qPCR) was performed using gene-specific primer pairs (Table S2 ), and the Roche SYBR-Green® master mix on an ABI7900HT Real-Time PCR System (Thermo Fisher Scientific Waltham, MA, USA). The transcripts were quantified using the following program: 3 min at 95 °C, 35 cycles of 15 s at 95 °C, 25 s at 60 °C, and 25 s at 72 °C. Values for each transcript were normalized to those of GAPDH using the 2−ΔΔCt method [18 (link)].
3
Quantification of miRNA and mRNA Expression from Blood and Tissues
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Total RNA was isolated from blood and tissues using the Hybride-R RNA Kit (GeneAll), according to the manufacturer’s instructions. cDNA for miRNA analysis was synthesized from 5 ng total RNA using the Universal cDNA synthesis kit II (Exiqon). For the determination of miRNA expression, qPCR was performed using the miRCURY LNA Universal RT micro RNA PCR LNAPCR primer sets for miR-16-5p, miR-130a-3p, miR-17-5p, miR-103-3p, miR-30c-5p, and miR-21a-5p (Exiqon), and the SYBR green PCR kit (Exiqon) in an ABI7900HT Real-Time PCR System (Thermo Fisher Scientific). cDNA for mRNA analysis was synthesized from 1 μg total RNA using PrimeScript 1st strand cDNA Synthesis Kit (Exiqon). For the determination of mRNA expression, qPCR was performed using gene-specific primer pairs and the Roche SYBR-Green master mix in an ABI7900HT Real-Time PCR System (Thermo Fisher Scientific).
4
Quantitative miRNA Expression Analysis
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The amount of miRNAs in tissues was determined by quantitative real-time PCR. Total RNA was isolated from tissues using the Hybride-R RNA Kit (GeneAll), according to the manufacturer’s instruction. cDNA for miRNA analysis was synthesized from 5 ng of total RNA using the Universal cDNA Synthesis Kit II (Exiqon). For the determination of miRNA expression by quantitative real-time PCR, the miRCURY LNA Universal RT micro RNA PCR LNA PCR primer sets for mmu-miR-126-5p, mmu-miR-466c-5p, mmu-miR-184-3p, and mmu-miR-200b-5p (Exiqon) and the SYBR Green PCR Kit (Exiqon) in an ABI7900HT Real-Time PCR System (Thermo Fisher Scientific) was used. The expression of miRNAs was determined by comparison of 5S rRNA expression in each sample.
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