M mlv reverse transcription kit

Total RNA of glioma tissues or cultured cells was isolated with Trizol reagent (Invitrogen, Carlsbad, CA, USA). Then the total RNA was reverse transcribed into cDNA using the M-MLV reverse transcription kit (Takara, Tokyo, Japan). QPCR was conducted using ABI 7300 Fast Real-time PCR System (Biosystems, Foster city, CA, USA) with a SYBR Premix Ex Taq kit (TaKaRa). The primer sequences were as follows:
SNHG5, 5′-CGAGTAGCCAGTGAAGATAATG-3′ (forward) and 5′-CACACAACA GTCAAGTAAACC-3′ (reverse); miR-1297, 5′-ACACTCCAGCTGGGTCCTTCATTCCA-3’ (forward) and 5′-GTGCAGGGTCCGAGGT-3′ (reverse); U6, 5′-CTCGCTTCGGCAGCACA-3’ (forward) and 5′-AACGCTTCACGAATTTGCGT-3’ (reverse); KPNA2, 5′-ATTGCAGGTGATGGCTCAGT-3′ (forward) and 5′-CTGCTCAACAGCATCTATCG-3′ (reverse); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-GCACCGTCAAGGCTGAGAAC-3’ (forward) and 5′-TGGTGAAGACGCCAGTGGA-3’ (reverse). GAPDH and U6 were employed as internal controls.

Total RNA was extracted from maize GSH99 using the TRIzol method and and then the first strand of cDNA was generated by using the M-MLV reverse transcription kit (Takara). Primers were designed using Primer 5.0. Primer pairs containing the BstE II (5’-ACTCTTGACCATGGTAGATCTGACGGATCGTA TCGTAGTACTAGTA-3’) and Bgl II (5’-GGGGAAATTCGAGCTGGTCACCAATAGTAAACCTCAGTTACTTG-3’) restriction enzyme sites were used. Subsequently, PCR was used to amplify the CDS amino acid sequence encoded by ZmMADS42. It was then inserted into the pCAMBIA3301 vector containing cauliflower mosaic virus (CaMV) 35S, and the constructed vector was introduced into Agrobacterium GV3101. The Agrobacterium-mediated technique was used to infect maize GSH99 callus tissue with an infection duration of 20 minutes, followed by a three-day dark incubation. Glyphosate was used for selection and culture, based on the properties of the pCAMBIA3301 vector. We proceeded with differentiation culture, rooting culture, acclimatization, and ultimately transplanting the seedlings.

Total RNA was isolated using TRIzol (Invitrogen). The RNA isolation was performed according to the manufacturer’s protocols. Reverse transcription of the RNA was performed using an M-MLV Reverse transcription kit (Takara). Real-time qPCR was performed using SYBR Premix Ex Taq (Takara) with a Step One Plus real-time PCR system (Life Technology). The results were analyzed using Expression Suite software (Life Technology) and StepOne 2.3 software. All data were normalized using endogenous β-actin as the control, and the 2^−ΔΔCt method was used to calculate the relative gene expression. Primers were purchased from Invitrogen and are listed in Table 1.

Cells were lysed and total RNA was isolated using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to manufacturer's protocol. RNA was reverse transcribed using the M-MLV reverse transcription kit (Takara, Tokyo, Japan). Quantitative PCR was performed using a SYBR Premix Ex Taq kit (Roche, Basel, Switzerland). The sequences of the primers obtained from Sangon Biotech Co., Ltd. were as follows: MKP-1 forward, 5-CCTTTCTGTACCTGGGCAGT-3 and reverse, 5-GGTTGGGACAATTGGCTGAG-3; GAPDH forward, 5-GGGTGTGAACCATGAGAAGT-3 and reverse, 5-GACTGTGGTCATGAGTCCT-3. GAPDH was used as an internal control. The thermocycling conditions for qPCR were as follows: 94°C for 2 min, followed by 40 cycles at 94°C for 15 sec, 60°C for 1 min and 72°C for 10 min. The 2^−ΔΔCq analysis method was used to calculate relative expression.