Total rna kit

Total RNA was extracted from ovarian tissue using a total RNA Kit (Ambion, Austin, TX, United States) in accordance with the manufacturer's instructions. RNA integrity and concentration were evaluated using NanoDrop (NanoDrop, Thermo Scientific) and Agilent 2100 Bio-analyzer (Agilent Technologies, Palo Alto, CA, United States). All the qualified RNA samples were then transported to OE Biotech. Co. (Shanghai, China) for transcriptome sequencing. The Illumina HiSeq 2500 system sequencing platform was used.

A total RNA kit (Invitrogen, Breda, Netherlands) was used to extract total tissue RNA, according to the kit instruction. The cDNA was synthesized by the avian myeloblastosis virus−mediated reverse transcription. Each gene primer was synthesized by Shanghai Sangon. The sequences of primers were as follows: LIMK1 (length: 138 bp): forward: 5′-GGGGCATCATCAAGAGCA-3′, reverse: 5′-GAGGACTAGGGTGGTTCAG-3′; destrin (length: 276 bp): forward: 5′-TGGTTGGAGATGTTGGTG-3′, reverse: 5′-TACAAGCCCGATTGAGAT-3′; cofilin1 (length: 120 bp): forward: 5′-CAAGAAGGCGGTGCTCT3-3′, reverse: 5′-ACAAAGGTGGCGTAGGG-3′; β-actin (length: 367 bp): forward: 5′-ACACTGTGCCCATCTACGAGGGG-3′, reverse: 5′-ATGATGGAGTTGAAG GTAGTTTCGTGGAT-3′. PCR products were electrophoresed on 2% agarose gel (ethidium bromide staining), and the Bio-Rad gel imaging system (Bio-Rad Laboratories, Inc., CA, USA) was used to photograph. AlphaImager 2200 software (Alpha Innotech Corporation, San Leandro, CA, USA) was used to scan and calculate the relative optical density on behalf of the abundance of gene expression, and β-actin expression abundance was considered as a reference to calculate the mean optical density value.

Leaves were collected, immediately frozen in liquid N₂ and stored at −80°C. A total RNA kit (Invitrogen) was used to isolate total RNA from the stored leaves, which was then treated with RNase-free DNase (Promega) to remove genomic DNA. M-MLV reverse transcriptase (Takara) was used to synthesize first-strand cDNA. A tomato ACTIN fragment amplified with Actin-RT primers was used as an internal control. The primer sequences are listed in Additional file 1: Table S1.

Total RNA was extracted from the tissue using the reagent box of Total RNA Kit (Invitrogen, Carlsbad, CA, US), according to the manufacturer's instructions. The concentration of RNA was measured by using a spectrophotometer and the purity was ascertained by the A 260/A 280 ratio with a Nanodrop® 8000. Total RNA from each sample was reverse transcribed to cDNA with an Omniscript® Reverse Transcription kit (Takara) with Oligo-dT primers (Takara) according to the manufacturer's instructions and used for RT-PCR. The target fragments were quantified by real-time PCR using a QuantiTectTMSYBR Green® PCR Kit (Roche) with 100 ng of the cDNA template. Each sample was tested in duplicate. The gene expression data were normalized to β-actin expression. The primers used correspond to the rat sequences shown in Table 1; primer design was done using Amplify software (TaKaRa, Nanjing, China). For each real-time PCR assay, the threshold cycle Ct was determined for each reaction. Ct values for each gene of interest were normalized to the housekeeping gene (β-action); PCR amplification efficiencies were taken into account by amplifying various amounts of target cDNA for each reaction. The fold differences in mRNA expression of samples were relative to the internal control sample, which was included in all runs.