Total RNAs were obtained from whole blood using the Hybrid-R™ Blood RNA purification kit (GeneALL, Seoul, South Korea) and then processed with DNase I to eliminate DNA contamination. NanoDrop was used to determine the quantity and quality of isolated RNA (Thermo Scientific, Wilmington, DE, United States). The cDNA synthesis kit (GeneALL) was used to produce complementary DNA (cDNA) in accordance with the manufacturer’s instructions. The cDNA was preserved at 20°C for additional investigation. Table 1 lists the primer sequences used in reverse transcription and quantitative polymerase chain reaction (qPCR) experiments. To normalize miRNA and mRNA levels, internal controls, such as U6 and ubiquitin C (UBC), were used. The qPCR was performed using the Step OnePlus™ Real-Time PCR and the RealQ Plus2x Master Mix (Ampliqon, Odense, Denmark). All qPCRs were accomplished in duplicate.
Step oneplus real time pcr
Manufactured by Ampliqon
Sourced in Denmark
The Step OnePlus™ Real-Time PCR is a compact, high-performance real-time PCR instrument designed for efficient and accurate gene expression analysis and quantification. It features a powerful optical system, intuitive software, and advanced temperature control for reliable and reproducible results.
Automatically generated - may contain errors
4 protocols using step oneplus real time pcr
1
Quantitative Analysis of Blood RNA
2
Quantifying RNA Expression Levels
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Total RNA was isolated from whole blood utilizing the Hybrid-R™ Blood RNA purification kit according to the manufacturer’s instructions (GeneALL, Seoul, South Korea) and treated with DNase I to remove DNA contamination. NanoDrop was employed to assess the quantity and quality of extracted RNA (Thermo Scientific, Wilmington, DE, United States). The synthesis of cDNA was done by the cDNA synthesis Kit (GeneALL) according to the manufacturer’s guidelines. The cDNA was stored at −20°C for further investigation. The primer sequences used in reverse transcription, as well as qPCR reactions, are listed in Table 1 . U6 and Ubiquitin C (UBC) were employed as internal controls to normalize miRNA and mRNA levels, respectively. The Step OnePlus™ Real-Time PCR and the RealQ Plus2x Master Mix (Ampliqon, Odense, Denmark) were used for the qPCR. All qPCR reactions were performed in duplicate.
3
Whole Blood RNA Extraction and qPCR Analysis
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Total RNA extraction was achieved from whole blood using a Hybrid-R™ Blood RNA purification kit (GeneALL, Seoul, South Korea) according to the manufacturer's protocol. The extracted RNA's concentration and quality were assessed with Nanodrop (Thermo Scientific, Wilmington, DE). cDNA was synthesized using the HyperScript™ kit (GeneAll) according to the manufacturer's instructions. Prepared cDNA was stored at -20 °C for further use. The primers were designed by Oligo7 software. The used primers for ERMN, LTN1, and ubiquitin C (UBC) as the housekeeping gene are shown in Table 1. UBC was chosen because it has been introduced as one of the most stable housekeeping genes in schizophrenia studies (Weickert et al. 2010; Silberberg et al. 2009 ). The qPCR was carried out by the Step OnePlus™ Real-Time PCR and the RealQ Plus2x Master Mix (Ampliqon, Odense, Denmark).
4
Blood RNA Extraction and qPCR Analysis
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Total RNA was extracted from whole blood based on the manufacturer's procedure using the Hybrid-R™ Blood RNA purification kit (GeneALL, Seoul, South Korea) and treated with DNase I to remove DNA contamination. NanoDrop was used to determine the amount and quality of isolated RNA (Thermo Scientific, Wilmington, DE). The synthesis of cDNA was done by the cDNA synthesis Kit (GeneALL) according to the manufacturer's instructions. The cDNA was kept at -20 °C for further investigation. Table 1 contains the primer sequences utilized in reverse transcription and quantitative polymerase chain reaction (qPCR) reactions. Internal controls hypoxanthine phosphoribosyl transferase 1 (HPRT1) and U6 were used to normalize mRNA and miR levels, respectively. HPRT1 was chosen because it has been demonstrated as a suitable reference gene for SCZ studies (Hwang et al. 2013; Chaumette et al. 2019; Asadzadeh Manjili et al. 2018) . The qPCR was performed using the Step OnePlus™ Real-Time PCR and the RealQ Plus2x Master Mix (Ampliqon, Odense, Denmark).
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