Cell lysates were collected and total RNA was extracted with RNase easy mini kit (Qiagen, USA). cDNA was generated from mRNA with oligo(dT)12-18 and Superscript IIITM reverse transcriptase (Gibco Invitrogen, USA) according to manufacturer’s protocol. The mRNA expression of target genes was quantified by using Applied Biosystem 7500 Real Time PCR System (Applied Biosystem, USA). Primer sequences and thermal cycles for detection of influenza virus matrix gene (M gene) and β-actin gene have been described previously57 (link). The expression of other target genes was quantified and normalized with the expression level of β-actin as described previously8 (link)55 (link)56 (link).
Rnase easy mini kit
Manufactured by Qiagen
Sourced in United States
The RNase Easy Mini Kit is a laboratory product designed for the efficient extraction and purification of RNA from a variety of sample types. It utilizes a silica-based spin column technology to capture and purify RNA, while removing contaminating DNA and other impurities.
Automatically generated - may contain errors
Lab products found in correlation
Applied biosystem 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Superscript iiitm reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Biophotometer, by Eppendorf (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Rnase free water, by Qiagen (1 mentions)
Rnase easy fibrous mini kit, by Qiagen (1 mentions)
5 protocols using rnase easy mini kit
1
Quantitative Real-Time PCR Analysis of Gene Expression
2
Transcription Start Site Mapping in V. vulnificus
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RNA was isolated from V. vulnificus using the RNase Easy Mini Kit (Qiagen, Valencia, CA), and RNA concentration was determined using a Biophotometer (Eppendorf, Hamburg, Germany). A 500 ng sample of RNA extracted from V. vulnificus was incubated with 5′-labeled primer RyhB-PE at 65 °C and chilled on ice. Reverse transcription was performed using a PrimeScript RT reagent kit (Takara, Tokyo, Japan). The resulting product and sequencing ladder were resolved on a 6% polyacrylamide sequencing gel to identify the transcription start site.
3
RNA Isolation and cDNA Synthesis
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Total RNA was isolated using RNAse easy mini kit (Qiagen). cDNA was synthesized using mRNA as a template and QuantiTect reverse transcription kit (Qiagen).
4
RNA Extraction from Spleen and Heart
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At harvest spleen or half heart was collected and stored at −80°C for RNA isolation. Spleens and hearts were homogenized and lysed using a Tissuelyser (Qiagen) with 7 mm stainless steel beads in RTL buffer with 0.5% DX buffer to reduce foam (Hilden, Germany). The homogenate was placed in an automated RNA isolation and purification instrument, QIAcube, with reagents for RNase Easy Mini Kit with a DNase step for spleen and cells or RNase Easy Fibrous Mini Kit including a DNase and proteinase K step for heart tissue (Qiagen). Spleen RNA was eluted into 30 μL and heart RNA into 60 μL RNase free water (Qiagen). RNA quantification was determined in μg/μL using NanoDrop (Thermo Scientific, Waltham, MA).
5
Harvesting and RNA Isolation from HEK 293 Cells
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Cultured HEK 293 cells from 10 cm dishes (1 mL/1 × 106 cells) were harvested in 2 mL epi tubes. Harvested cells were then washed with PBS and total RNA was isolated using RNase easy mini Kit (Qiagen, Germantown, MD, USA) as per the instruction of the manufacturer.
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