Primescript rt reagent kit perfect

Frankia cells were acclimated to N- conditions as described above for ARA measurements. After 4 d in BAP-TN- medium, cells were collected by centrifugation (2,500×g, 20°C, 10 min) and total RNA was purified by the cetyltrimethylammonium bromide (CTAB) method as described previously (14 (link)). Contaminating DNA was removed by a treatment with the TURBO DNA-free kit (Thermo Fisher Scientific). cDNA was synthesized using a PrimeScript RT reagent Kit (Perfect Real Time; Takara Bio, Ohtsu, Japan). Briefly, a 20-μL reaction mixture contained 2 μg total RNA and 2 pmol gene-specific reverse primers (Table S1). The reverse transcription reaction was incubated at 42°C for 15 min, followed by an incubation at 50°C for 15 min. Real-time PCR was performed using the TaqMan Gene Expression Master Mix (Applied Biosystems, CA, USA) and StepOnePlus Real Time PCR System (Applied Biosystems). The reaction mixture contained 2 pmol gene-specific forward and reverse primers (Table S1), 1.5 pmol TaqMan probe (Table S1), and cDNA derived from 0.3 ng (16S rRNA, internal standard) or 100 ng (nifE, nifH, and nifV) total RNA in a total volume of 10 μL.

Following 24 and 48 h of incubation of the BY318 strains (BY318-Control, BY318-opt_ecoFBPase, and BY318-ATPase) and 48 h of incubation of the YHI030 strains (YHI030-Control, YHI030-opt_ecoFBPase, and YHI030-ATPase), the cells were harvested at OD₆₀₀ = 3.0 after 48 and 72 h of incubation, respectively. The cells were centrifuged at 17,970 × g for 3 min and washed with TE buffer. After discarding the supernatant, 200 μL RNA buffer (0.8 M sorbitol, 100 mM EDTA, 14 mM β-mercaptoethanol) and zymolyase-20 T (1000 U/mL) (Nacalai Tesque, Kyoto, Japan) were added to the culture, which was incubated at 30 °C for 30 min. The purified RNA was subjected to cDNA synthesis using the PrimeScript™ RT Reagent Kit (Perfect Real Time, TaKaRa). The target genes were quantified using TB Green™ Premix Ex Taq^™ (Tli RNaseH Plus, TaKaRa) and a StepOnePlus real-time PCR system. Amplification was performed using the following primer sets: ACT1_for_RT-PCR_F/ACT1_for_RT-PCR_R for ACT1 (control gene), ATPase_for_RT-PCR_F/ATPase_for_RT-PCR_R for ATPase, and optFBPase_for_RT-PCR_F/optFBPase_for_RT-PCR_R for FBPase. Target genes were quantified based on the obtained amplification signals.

The total RNA of tomato leaves was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, United States), following the manufacturer’s protocol. RNA was then precipitated by adding one volume of 6 M LiCl and keeping it on ice for 4 h. Afterward the pellet was washed using 3 M LiCl and was dissolved in RNase-free water. Finally, to remove any contaminating genomic DNA, 2 U of TURBO DNase (Ambion, Austin, TX, United States) were added per microliter of RNA. For the quantitative RT-PCR (qRT-PCR) analysis, one microgram of total RNA was employed to obtain the corresponding cDNA target sequences using an oligo(dT)18 primer and the PrimeScript RT reagent kit (Perfect Real Time, Takara Bio Inc., Otsu, Shiga, Japan), following the manufacturer’s directions. Quantitative PCR was carried out as previously described [81 (link)]. A housekeeping gene transcript, actin or elongation factor 2, was used as the endogenous reference. The PCR primers were designed using the online service Primer3 (https://primer3.ut.ee/) and are listed in Table S1.

One microgram of total RNA was reverse transcribed into cDNA in a final reaction volume of 10 µl using the PrimeScript™ RT reagent kit (Perfect Real Time, TaKaRa, Dalian, China) or the miRcute miRNA first-strand cDNA synthesis kit (TIANGEN Biotech Co., Ltd. Beijing, China). The resulting cDNAs were diluted 20-fold in nuclease-free water (Invitrogen) and were used as templates for qPCR. qPCR was performed in 20 µl reactions containing 10 µl of 2× SYBR Premix EX Taq™ (TaKaRa) or 10 µl of 2× miRcute miRNA premix (TIANGEN), 2 µl of cDNA, and 300 nM of gene-specific primers (Table 1). qPCR was performed using the 7500 Real-Time PCR Detection System (Applied Biosystems, Foster, CA). The efficiency of the reaction was measured with primers using serial dilutions of the cDNA (1∶1, 1∶5, 1∶25, 1∶125, 1∶625 and 1∶3,125). Each sample was tested in triplicate. The relative mRNA expression was analyzed using the Pfaffl method [22] (link). The data were normalized to the expression of GAPDH and are expressed as fold changes relative to untreated controls.

Pseudogulbenkiania sp. strain 2002 and strain NH8B were grown in basal medium (5 mL) supplemented with 10 mM acetate under oxic or anoxic conditions. When grown under anoxic conditions, 5 mM nitrate was added to this medium as an electron acceptor. Total RNA was extracted using the RiboPure Bacteria kit (Ambion). After the DNase treatment, complementary DNA (cDNA) was synthesized using random hexamers and a PrimeScript RT reagent kit (Perfect Real Time) (Takara Bio). The resulting cDNA was used to detect gene transcription by PCR (Table S2). While genomic DNA was used as a positive control, RNA samples were used as negative controls in order to verify the absence of contamination by genomic DNA. Experiments were performed in triplicate (i.e., three test tubes per condition for each strain).

To verify the effect of key genes on leaf traits, the individuals with extreme phenotype values of leaf lobe (LLo) and circularity (Cir) were selected as samples for RT–qPCR (Supplementary Data Table S1). In September 2020, 10–20 newly developed leaves were harvested from the eastern- or southernmost lateral branches, and the petioles were removed. The samples were immediately placed in liquid nitrogen and stored at −80°C for subsequent RNA extraction. Total RNA was isolated from samples by using an RNAprep Pure Plant Plus Kit (TIANGEN Biotech Co., Ltd., Beijing, China). The total RNA concentration and integrity were determined using a NanoDrop (Thermo Fisher Scientific Inc.). RNA was reverse-transcribed into cDNA using the PrimeScript™ RT Reagent Kit (Perfect Real Time; Takara Biomedical Technology Co., Ltd., Beijing, China). Primary pairs were designed for CRK2 and SRK. cDNA was amplified using a KAPA SYBR^® FAST Kit (Shanghai Roche Pharmaceutical Co., Ltd.), and the gene expression levels were calculated by the 2^−ΔΔCt method.