Plant total rna kit

Blueberry (Vaccinium corymbosum ‘Sierra’) fruits were harvested from an organic blueberry farm (Tianshuo Farm in Hebei Province, China) during the 2013 to 2015 growing season. The tissues at three different developmental stages including green fruit (G, 30d), pink fruit (P, 50d) and blue fruit (B, 75d), were randomly sampled in the field from 4- or 5-year-old healthy blueberry plants. The plants had been propagated by tissue culture, thus all came from the same mother plant. All samples intended for RNA extraction were flash-frozen in liquid nitrogen immediately after collection and stored at −80 °C until use. Fifty fruits were collected at each time point and combined for RNA extraction. RNA was extracted using the Plant total RNA Kit (TIANGEN, Beijing, China). The purified RNA quality and quantity was evaluated using a spectrophotometer (Thermo Scientific, Waltham, MA, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

After 6 days of culturing for the wild-type and mutant strains in different nitrogen sources, the total RNA of strains was extracted using the plant total RNA kit (Tiangen Biotech, Beijing, China), and cDNA was synthesized using a reverse transcription kit (TransGen Biotech, Beijing, China). Finally, the relative expression levels of UGT and GLS were quantitatively determined using β-actin as an internal reference by QPCR kit from Promega; the kit used SYBR green as the fluorescence label, and the expression levels were assessed using 2^-ΔΔCT.

For RNA extraction, we used the Plant Total RNA Kit (Tiangen, Beijing, China), and PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa, Kusatsu, Japan) was used for the synthesis of the first-strand cDNA. The ABI Step, One Plus instrument was used for qRT-PCR experiments. Experiments were repeated 3 times. The comparative 2−ΔΔCT (A method of qRT-PCR fluorescence quantitative data analysis, ΔΔCt = ΔCt experimental group − ΔCt control group) method was used for gene expression level analysis. For quantitative analysis, Exocyst complex component gene (Sec3A) was used as the internal reference. Reactions contained the following: 5 μL of 2x × TB Green Premix Ex Taq II, 1 μL of template cDNA, 0.4 μL of forward and Universal miRNA qPCR Primer, 0.2 μL of Passive Reference Dye and water to 10 μL. PCR amplification was performed as follows: 95 °C for 30 s, 40 cycles of 95 °C for 5 s, 58 °C for 30 s.