Dynabead mrna direct kit

Mouse oocytes maturated in vitro cultured for 12 h, and in vivo matured oocytes were obtained from ampullar potion. The oocytes in the IVO group and the IVM group were collected. Total RNA was extracted from exactly 30 oocytes with a Dynabead mRNA DIRECT kit (Invitrogen Dynal, Oslo, Norway). According to the manufacturer's instructions (Invitrogen), the first strand was synthesized with a cDNA synthesis kit (Takara) by Oligo (dT) 12–18 primers. The cDNA was stored at −20°C until analysis. The levels of relevant mRNAs were determined by quantitative RT-PCR using a FastStart Universal SYBR Green Master (Rox; Roche Applied Science, Mannheim, Germany) with One plus Real-Time PCR System (Applied Biosystems, Life Technologies, Carlsbad, CA, USA). Gene expression levels were analyzed using the 2–ΔΔCt method after the melting-curve analysis was completed. The expression levels of the target genes were then normalized to the expression level of GAPDH in each sample. The primers are listed in Supplementary Table 1.

The mRNA was extracted from cells isolated by FACS using the Dynabead mRNA Direct kit (Invitrogen). Whole aorta tissue was homogenized using a tissue-lyser machine (Qiagen). Briefly, samples were frozen with liquid nitrogen and were homogenized with the tissue-lysser for 2 min and frozen again in liquid nitrogen. This procedure was repeated four times. Whole aorta RNA was extracted using Tri Reagent (Sigma-Aldrich), followed by RNA purification with the RNeasy mini kit (Qiagen). Reverse transcription of 500 ng RNA was performed using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). QPCR experiments were conducted using SYBR Green (Applied Biosystems). Samples were loaded on a 384-well plate and analyzed on a 7900HT machine (Applied Biosystems). The expression level of each gene was determined using the relative standard curve method. The standard curve was performed using serial dilutions of mouse reference total RNA (Clontech). The expression level of each gene was calculated by interpolation from the standard curve. All values were normalized with Gapdh and Hprt1 as endogenous housekeeping genes. The sequences of the primer pairs used are listed in the Supplementary Table 1.

Real-time quantitative PCR and the ΔΔC_T method normalized by β-actin were employed to measure the KIF2A gene expression. Each sample contained 50 oocytes, and total RNA was extracted by a Dynabead mRNA DIRECT kit (Life Technologies AS, Oslo, Norway). A cDNA synthesis kit (Toyobo, Osaka, Japan) was used to generate the first strand cDNA with Oligo (dT) 12–18 nucleotide primers (Takara Bio Inc., Tokyo, Japan). The following primers were used to amplify the KIF2A and β-actin cDNA fragments:
KIF2A forward, 5′-GACCTGGCTGGGAACGAAAG-3′
KIF2A reverse, 5′- TTTGTTTCTACCTAAGGCTCGGATG-3′
β-actin forward, 5′-CATCCGTAAAGACCTCTATGCCAAC-3′
β-actin reverse, 5′- ATGGAGCCACCGATCCACA-3′
The SYBR Green real-time PCR Master Mix kits (Life Technologies, Carlsbad, CA, USA) was utilized with a Step One real-time PCR system (Applied Biosystems, Foster City, CA, USA). The following conditions were used to perform the process: 50 °C for 2 min and 95 °C for 2 min; followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.

Porcine OCT4, CDX2, or NANOG gene expression levels were analyzed by real-time quantitative (q)PCR using the ΔΔCT method[28 (link)]. Total RNA was extracted from 50 oocytes using a Dynabead mRNA DIRECT Kit (Life Technologies; Foster City, CA, USA). First-strand cDNA synthesis was completed using a cDNA Synthesis Kit (Takara; Kyoto, Japan) and oligo(dT) 12–18 primers. The PCR primers used to amplify OCT4, CDX2, and NANOG genes are listed in S1 Table. Real-time PCR was performed with SYBR Green in a final reaction volume of 20 μL (qPCR kit, Finnzymes; Vantaa, Finland). PCR conditions were as follows: 95°C for 3 min, followed by 39 cycles of 95°C for 15 s, 57°C for 15 s, 72°C for 45 s, and a final extension of 72°C for 5 min. Finally, relative gene expression levels were quantified by normalizing to the respective GAPDH mRNA level. Experiments were conducted in triplicate.

Analysis of Axin1 gene mRNA expression was measured by real-time quantitative PCR and the ΔΔC_T method. We used a Dynabead mRNA DIRECT kit (Life Technologies AS, Oslo, Norway) to extract the total RNA from 70 oocytes. First strand cDNA was generated using a cDNA synthesis kit (Toyobo, Tokyo, Japan), using Oligo(dT) 12–18 nucleotide primers (Takara Bio Inc., Tokyo, Japan). A cDNA fragment of Axin1 was amplified using the following primers: forward, CAC CCA GAA GCT GCT ATT GGA GA, reverse, CCA GGG CAT AGC CAG AGT TGA. We used SYBR Green real-time PCR Master Mix kits (Life Technologies, Carlsbad, USA) with a Step One real-time PCR system (Applied Biosystems, Foster City, USA) under the following conditions: 50°C for 2 min and 95°C for 2 min; followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.

Tmod3 expression was analyzed by real-time quantitative PCR (qRT-PCR) and the ΔΔCT method62 (link). Total RNA was extracted from 30 oocytes using a Dynabead mRNA DIRECT Kit (Life Technologies, Foster City, CA). First-strand cDNA was generated using a cDNA Synthesis Kit (Takara, Kyoto, Japan) and Oligo (dT)_12–18 primers. The PCR primers used to amplify Tmod3 are listed in Table 1. qRT-PCR was performed with SYBR Green in a final reaction volume of 20 μl (qPCR kit, Finnzymes, Vantaa, Finland). PCR conditions were as follows: 94 °C for 10 min, followed by 39 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 45 s, with a final extension step at 72 °C for 5 min. Finally, relative gene expression was quantified by normalization to the level of GAPDH mRNA. Experiments were conducted in triplicate.