Extraction of total RNA was performed using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. RNA integrity and quantity were evaluated via gel electrophoresis and spectrophotometry (NanoDrop 2000, Thermo Scientific), respectively. For cDNAs synthesis from total RNA, the PrimeScript™ 1st Strand cDNA Synthesis Kit (TaKaRa Bio, Japan) was used. The sequence of circ_0047303 was obtained from the CircInteractome database, and specific primers were designed and used in real‐time PCR (Table S1 ). Quantitative real‐time PCR was carried out using RealQ Plus 2x Master Mix Green low Rox (Ampliqon) by Light‐Cycler96 Roche thermocycler. All reactions were performed in 10 µl and duplicated. Gene expression was quantitated using the 2−ΔΔCt method and normalized to the PUM1 gene as the internal control. PCR products were analysed by melting curve analysis and agarose gel electrophoresis. The circular junction of circ_0047303 was confirmed by Sanger sequencing.
Realq plus 2x master mix green low rox
Manufactured by Ampliqon
Sourced in Denmark
RealQ Plus 2x Master Mix Green Low ROX is a ready-to-use master mix for real-time PCR applications. It contains all the necessary components for efficient and sensitive DNA amplification, including a DNA polymerase, dNTPs, and a low concentration of the ROX passive reference dye.
Automatically generated - may contain errors
Lab products found in correlation
Abi prism 7500 sequence detection system, by Thermo Fisher Scientific (2 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Primescript 1st strand cdna synthesis kit, by Takara Bio (1 mentions)
Lightcycler 96, by Roche (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Rnx plus, by CinnaGen (1 mentions)
4 protocols using realq plus 2x master mix green low rox
1
Circular RNA circ_0047303 Quantification
2
Antioxidant Gene Expression in Expanded Blastocysts
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Total RNA was extracted from 3 pools of 15 expanded blastocysts per group, using the
RNeasy Micro Kit (Qiagen, Hilden, Germany) following the manufacturer instructions.
First-strand cDNA synthesis was carried out using the QuantiTect Reverse Transcription Kit
(Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Real-time reverse
transcription - polymerase chain reaction (RT-PCR) was performed using an ABI Prism 7500
Sequence Detection System (Applied Biosystems, Foster City, USA). The PCR amplification
was conducted in a final volume of 25 μl consisting of 1 μl of the cDNA template, 12.5 μl
of RealQ Plus 2x Master Mix Green Low ROX (Ampliqon A/S, Odense, Denmark), and 1 μl of
each primer (10 pmol/μl). Glyceraldehyde-3-phosphate dehydrogenase
(Gapdh) was used as a reference (22 (link)). The gene expression of
GPx1, Sod1 and Cat as main
antioxidant enzymes, and Bcl2l1 and Bax in expanded
blastocysts, was analyzed using the 2-∆∆Ct method. The primers used for RT-PCR
are listed in Table 1.
RNeasy Micro Kit (Qiagen, Hilden, Germany) following the manufacturer instructions.
First-strand cDNA synthesis was carried out using the QuantiTect Reverse Transcription Kit
(Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Real-time reverse
transcription - polymerase chain reaction (RT-PCR) was performed using an ABI Prism 7500
Sequence Detection System (Applied Biosystems, Foster City, USA). The PCR amplification
was conducted in a final volume of 25 μl consisting of 1 μl of the cDNA template, 12.5 μl
of RealQ Plus 2x Master Mix Green Low ROX (Ampliqon A/S, Odense, Denmark), and 1 μl of
each primer (10 pmol/μl). Glyceraldehyde-3-phosphate dehydrogenase
(Gapdh) was used as a reference (22 (link)). The gene expression of
GPx1, Sod1 and Cat as main
antioxidant enzymes, and Bcl2l1 and Bax in expanded
blastocysts, was analyzed using the 2-∆∆Ct method. The primers used for RT-PCR
are listed in Table 1.
3
SYBR Green Real-Time PCR Assay
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Gene expression level was measured by SYBR green-based Real-Time PCR using a thermocycler (Rotor-Gene 6000, Bosch, Qiagen, Germany). The qRT-PCR assay was accomplished in 15 µl final reaction volume using 1.5 μL template target cDNA, 1.2 μL forward and reverse primer, 7.5 μL RealQ Plus 2x Master Mix Green- Low ROX (Ampliqon, Denmark), and 4.8 μL water to reach total volume. Primers were designed via oligo 7/56 software and their specificity was confirmed in NCBI Blast database. The primers pairs for the mention genes are available in table 1 . Thermal cycling for each reaction (HIF1α, VEGF-A, and ABL) included an initial hold at 95°C for 10 minutes followed by 40 denaturation cycles at 95°C for 10 seconds and annealing/extension at 58°C (VEGF-A), 59°C (HIF1α) and 65°C (ABL) for 20 seconds. All the reactions were done in triplicated manner. A standard curve was obtained by four consecutive 1:10 dilutions of a positive sample (1, 0/1, 0/01 and 0/001). Expression level of target genes analyzed in comparison with ABL (housekeeping gene) by Livak method (2-ΔΔct) (Livak and Schmittgen, 2001; Schmittgen and Livak, 2008).
4
Antioxidant and Apoptosis Regulation in Ovaries
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Total RNA was extracted from ovaries using the RNX plus (Cinnagen, Iran) following the manufacturer's instructions. First‐strand cDNA synthesis was carried out using the QuantiTect Reverse Transcription Kit (Qiagen, Germany) according to the manufacturer's instructions. Real‐time RT‐PCR was performed using an ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The PCR amplification was carried out in a final volume of 25 μl, including 1 μl of the cDNA template, 1 μl of each primer (10 pmol/μl) and 12.5 μl of RealQ Plus 2x Master Mix Green Low ROX (Ampliqon, Odense, Denmark). Beta‐actin (Actb) was used as a reference gene for normalization of the target gene dosage level. Genes expression of glutathione peroxidase 1 (GPx1), superoxide dismutase 1 (Sod1) and catalase (Cat) as main antioxidant enzymes, and Bcl2 (apoptotic inhibitor) and Bax (apoptotic activator) in ovaries were analysed. Samples were analysed using the 2−∆∆Ct method. The primers used for RT‐PCR are listed in Table 1 .
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