RNA of MIN6 cells and RNA of isolated islets from congenic (Nidd13/NZO; B/B, N/N) and parental NZO mice was reversed transcribed (M-MVL RT, Promega, Madison, WI, USA) for quantitative real-time PCR. Genes of interest were detected using specific TaqMan (Thermo Fisher Scientific, Waltham, MA, USA) and IDT (Integrated DNA Technologies, Coralville, IA, USA) probes. Expression levels were evaluated using the 2(-Delta C(T)) method [31 (link)] with β-actin (Actb), eukaryotic translation elongator factor 2 (Eef2), or TATA box binding protein (Tbp) as internal controls.
M mvl rt
Manufactured by Promega
Sourced in United States
The M-MVL RT is a reverse transcriptase enzyme used for the conversion of RNA to cDNA in a variety of molecular biology applications. It is a thermostable enzyme with robust activity and fidelity.
Automatically generated - may contain errors
4 protocols using m mvl rt
1
Quantitative Real-Time PCR Analysis of MIN6 Cells and Islets
2
Total RNA Extraction and Real-Time PCR Analysis
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Total RNA was extracted using Easy Blue® reagent (Intron Biotechnology, Gyeonggi-do, South Korea) and following the chloroform/isopropanol purification procedure. cDNA synthesis was performed with 1 μg total RNA using Moloney murine leukemia virus reverse transcriptase (M-MVL RT) (Promega, Madison, WI, USA) and random primers according to the manufacturer’s instructions. For real-time PCR analysis, endogenous mRNA levels were measured based on SYBR Green (Applied Biosystems, Foster City, CA, USA) detection with an ABI Prism 7500 machine (Applied Biosystems). Results were normalized with the β-actin expression. The real-time PCR primers used are shown in Table S1 .
3
Transcriptome Analysis of Differentiated MSCs
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RNA of differentiated MSCs was isolated (miRNeasy Micro Kit, Qiagen, Hilden, Germany) and reversed transcribed (M-MVL RT, Promega, Madison, WI, USA) for quantitative real-time PCR. Genes of interest were detected using specific TaqMan (Thermo Fisher Scientific, Waltham, MA, USA) and IDT (Integrated DNA Technologies, Coralville, IA, USA) probes. Expression levels were evaluated using the 2(-Delta CT) method [4 (link)] with eukaryotic translation elongator factor 2 (Eef2) as internal control.
For RNA sequencing, isolated MSCs were expanded for 4 days and RNA was isolated according to the manufacturer’s protocol (miRNeasy Micro Kit, Qiagen, Hilden, Germany).
For RNA sequencing, isolated MSCs were expanded for 4 days and RNA was isolated according to the manufacturer’s protocol (miRNeasy Micro Kit, Qiagen, Hilden, Germany).
4
Reverse Transcription and PCR Analysis
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Total RNA and 0.5 pg oligo (dT) primer were mixed and incubated at 70 8C for 10 min; cDNA was then synthesized according to the manufacturer's protocol. The reverse transcription reaction was (in brief) carried out in a final volume of 20 ml M-MVL RT 5X buffer containing 0.5 mM dNTPs and 200 U of M-MLV RT (Promega). The reaction mixture was incubated at 42 8C for 90 min and then at 94 8C for 2 min. cDNA equivalent to that obtained from one oocyte served as a template for PCR analysis. The PCR primers used are listed in Table 1. Following PCR, 20-ml aliquots of each sample were resolved using electrophoresis on a 1.5% agarose gel, and the results were recorded using an Image Analyzer.
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