To verify the accuracy and repeatability of the RNA-seq data, 15 DEGs were selected for qRT-PCR validation. The total RNA was extracted from the first true leaf on the 14th d after Pi deficiency treatment by using RNAprep Pure Plant Plus Kit (Tiangen Biotech, Beijing, China), and cDNA was synthesized using PrimeScriptTM RT Master Mix. Quantitative RT-PCR was performed using an SYBR® Premix Ex TaqTM Kit (Tiangen Biotech, Beijing, China) with a Roche LightCycler 96 real-time PCR machine (Roche, Basel, Switzerland) with six replicates. The relative expression was calculated using the 2−∆∆Ct method [58 (link)]. Actin was used as an internal control [59 (link)]. The primers used for qRT-PCR were listed in Supplementary Table S1 .
Primescript rt master mix
Manufactured by Tiangen Biotech
Sourced in China
PrimeScript RT Master Mix is a ready-to-use solution for reverse transcription (RT) reactions. It is designed for the efficient conversion of RNA into cDNA, which is a key step in various molecular biology applications, such as gene expression analysis and RT-PCR.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Sybr premix ex taqtm kit, by Tiangen Biotech (1 mentions)
Rnaprep pure plant plus kit, by Tiangen Biotech (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium dmem medium, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Ultrapure rna kit, by Tiangen Biotech (1 mentions)
5 protocols using primescript rt master mix
1
Validation of RNA-seq Data by qRT-PCR
2
Regulation of Angiogenesis by miR-424 in Endothelial Cells
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Dulbecco's modified eagle medium (DMEM) medium, Roswell Park Memorial Institute (RPMI) 1640 medium, and LipofectamineTM 2000 were purchased from Invitrogen, USA; fetal bovine serum, penicillin, streptomyces, trypsin, and Radio Immunoprecipitation Assay (RIPA) Lysis Buffer were purchased from Beijing Solabao Technology Co., Ltd. (Beijing, China); propranolol hydrochloride was purchased from Shanghai Latin Life Technology Co., Ltd. (Shanghai, China); TransZol Up Plus RNA Kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd. (Beijing, China); PrimeScriptTM RT Master Mix was purchased from Tiangen Biochemical Technology (Beijing, China); Negative Control (NC) inhibitor, NC mimic, miR-424 inhibitor, and miR-424 mimic was purchased from Shanghai Abbots Biotechnology Co., Ltd. (Shanghai, China); primers were synthesized by Guangzhou Kinco Biotechnology Co., Ltd. (Guangzhou, China); TaqMan MicroRNA was purchased from Thermofisher, USA; specific primary antibodies [vascular endothelial growth factor-A (VEGFA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] and secondary antibodies immunoglobulin-G (IgG) were purchased from Shanghai Abkang Trading Co., Ltd. (Shanghai, China); crystal violet, 4% paraformaldehyde, Cell counting kit-8 (CCK-8) Kit, and Annexin V-FITC/PI Kit were purchased from Shanghai Biyuntian Biotechnology Co., Ltd.(Shanghai, China).
3
Transcriptome Profiling of Melon Development
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Total RNA was extracted with an ultrapure RNA Kit (Tiangen, Beijing, China) and template cDNA synthesis was performed using the PrimeScript™ RT Master Mix (Tiangen, Beijing, China) following the manufacturer’s instructions. A part of the template cDNA was used for RNA-Seq. Meanwhile, the template cDNAs amplified with a 20 μL of reaction solution by using TOROGreen® qPCR Master Mix (Toroivd, Shanghai, China). The qRT-PCR program consisted of a preliminary step of 60 s at 95 °C, followed by 40 cycles at 95 °C for 10 s, and 60 °C for 30 s. The Actin7 from melon was used as an internal control [55 (link)]. The relative gene expression level was calculated via the 2−ΔΔCt method. Three biological and three technical replicates were used for each sample. All the primers used in this study are listed in Table S2 . In addition, the raw transcriptome sequencing data was used to analyze the tissue specificity of NF-Y members and their roles in fruit ripening, including five melon plant tissues, including roots, leaves, male flowers, female flowers, and fruits (PRJNA383830) [62 (link)], as well as fruit development of green-fleshed (Dulce) and orange-fleshed (Tam-Dew) at 10, 20, and 30 days after anthesis (DAA) and maturity, respectively (PRJNA286120) [37 (link)].
4
Quantifying mRNA Expression by qRT-PCR
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Total RNA was extracted from cells by TRIzol reagent (Invitrogen, NY, USA). cDNAs were generated from RNA via reverse transcription using PrimeScript RT Master Mix (TIANGEN, Beijing, China). The primers for GAPDH were as follows: Forward 5'-GTCTCCTCTGAC TTCAACAGCG-3' and Reverse 5'-ACCACCCTGTTG CTGTAGCCAA-3'. The primers for B3GNT3 were Forward 5'-TATGTGTCTGGAGCTTGAGG-3', Reverse 5'-AAGGATGTGTAGGAGTTCGC-3'. qRT-PCR was performed using the SYBR Premix Ex Taq reaction system according to the manufacturer’s instructions. The levels of mRNA were measure by calculating 2−ΔΔCT.
5
COP1 mRNA Expression in Gastric Tissues
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Trizol reagent (Thermo Fisher Scientific, USA) was adopted for extraction of total RNA from GC and adjacent gastric tissues according to the manufacturer's instructions, with the reverse transcription process performed via the PrimeScript RT Master Mix (Tiangen, Beijing, China). Thereafter, the expression of COP1 mRNA of tissues was examined using SYBR Green qPCR Master Mix (MCE, Shanghai, China), with 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds. The primers were purchased from Sangon Biotech, China, and the sequences were as follows:
COP1-F, 5′-CTGTTTGGGAGGTCGGGTAAATGG-3′
COP1-R, 5′-AGTGGTGTGAGTGAGAGGCTGAG-3′.
COP1-F, 5′-CTGTTTGGGAGGTCGGGTAAATGG-3′
COP1-R, 5′-AGTGGTGTGAGTGAGAGGCTGAG-3′.
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