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32 protocols using All-In-One 5X RT MasterMix

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Quantitative Detection of 'Candidatus Liberibacter asiaticus'

For CLas detection, total DNA was extracted from midrib sections of sugar orange plants using Biospin Omini Plant Genomic DNA Extraction Kit (BioFlux, Hangzhou, China). The CLas detection was conducted by both PCR and quantitative PCR (qPCR). The PCR was performed using 16S rDNA primer OI1/OI2c with 2X Taq Master Mix (Vazyme, Nanjing, China) in a 20 μL reaction system. The bacterial populations (CLas cells μg-1 of citrus DNA) were quantified with qPCR assay that was described by Du M. et al. (2021) (link). In the qPCR assay, CLasgyrA of CLas was detected, and 18S rRNA of citrus was used as the internal reference. BlastTaq™ 2X qPCR MasterMix (abm, Canada) was used for qPCR amplification. Primer pairs are listed in Table S1.
Total RNA was extracted from plant tissues and psyllids using RNAiso Plus (Takara, Japan). In a 20-μL volume, 1μg of total RNA was reverse transcribed with All-In-One 5X RT MasterMix (abm) following the manufacturer’s instructions. Quantitative RT-PCR (qRT-PCR) assays were conducted to analyze gene transcript level. Primer pairs of selected genes are listed in Table S1. The genes NbACTIN and CsGAPDH were used as endogenous controls. The qRT-PCR analyses were performed in a 10 μL reaction system. The 2-ΔΔCt method was used for the determination of relative gene transcription (Livak and Schmittgen, 2001 (link)).
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Total RNA from HL-1 cells was extracted using the TRIzol Universal Reagent (TIANGEN, Beijing, China) following the manufacturer’s instructions. The total RNA concentration of each sample was then measured using nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). Next, the complementary DNA (cDNA) was synthesized by using the All-In-One 5X RT MasterMix (ABM, G490, USA). The procedure was conducted following the manufacturer’s protocol. In short, the reaction was executed at 25 °C for 10 min and then 42 °C for 15 min, followed by 5 min at 85 °C. Quantitative analysis of RNA was accomplished using the Roche LightCycler 480 (Roche, Basel, Switzerland) with BlasTaq™ 2X qPCR MasterMix (G891; ABM, New York, NY, USA). The reaction conditions were as follows: The reaction was executed at 95 °C for 3 min, followed by 40 cycles of 15 s at 95 °C and 60 s at 60 °C. The relative quantification of the target genes was normalized by GAPDH levels and calculated using the 2−ΔΔCT method. The primer sequences were presented in Table 1.
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Total RNA was extracted from shoot apices of 6-week-old plants using RNAzol RT RNA Isolation Reagent (MRC, Cat: RN190). Plant materials were thoroughly ground using the Tissuelyser-48 (Shanghai Jingxin). Two-microgram RNA samples were subjected to reverse transcription using the All-In-One 5X RT MasterMix (abm, Cat: G592). Quantitative real-time PCR analysis was conducted on a Bio-Rad CFX Connect real-time PCR detection system (CFX96, Bio-Rad) using FastStart Universal SYBR Green Master (Rox) reagent (Roche, Cat: 4913850001). The relative transcript level was calculated using the 2−ΔΔCt method, with MtUBIQUITIN (Medtr3g110110) serving as the internal control. Each reaction was conducted in triplicate. The primers used are listed in Supplementary Data 1.
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RNA was isolated from cells grown in SC media by standard acid phenol purification as previously described (56 (link)). ABM All-In-One 5X RT MasterMix (ABM) was used to generate cDNA. Primers for gene expression analysis are indicated in Table S4. A minimum of three biological replicates, as well as three technical replicates, were performed for each biological replicate using the comparative CT method (2−ΔΔCT) see Table S5.
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Cells were grown to mid-log phase (OD600 ~ 0.8) in YPD and, where indicated, treated with 50 ng/ml micafungin for 1 h prior to RNA extraction. RNA was isolated and purified using acid phenol as described (Serratore et al., 2018 (link); Baker et al., 2022 (link)). Reverse transcription was performed using All-in-One 5X RT MasterMix (ABM). Primer Express 3.0 software was used to design primers and qRT-PCR was performed as previously described (Serratore et al., 2018 (link); Baker et al., 2022 (link)) with at least three biological replicates. Data were analyzed using the comparative CT method (2-ΔΔCT). Internal controls were RDN18 (18S rRNA) for C. albicans and ACT1 for S. cerevisiae. All samples were normalized to an untreated wild-type strain. Primers used for all qRT-PCR analyses are listed in Supplementary Table S3.
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RNA was isolated from cells grown in SC media by standard acid phenol purification as previously described (37 (link)). Reverse transcription to generate cDNA was performed using the ABM All-In-One 5X RT MasterMix (ABM). Primer Express 3.0 software was used for designing primers for gene expression analysis by quantitative real-time polymerase chain reaction (Table S4). A minimum of three biological replicates, as well as three technical replicates, were performed for each biological replicate. All data were analyzed using the comparative CT method (2−ΔΔCT). RDN18 (18S ribosomal RNA) was used as an internal control. All samples were normalized to the Cg2001 WT strain (Table S5).
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Total RNA was isolated using TRIzol reagent (SparkJade, Harbin, China) according to the standard protocol. Then, the genomic DNA removal and cDNA synthesis were performed using All-In-One 5X RT MasterMix (abm, Zhenjiang, China). Primers used for qRT-PCR were designed by Integrated DNA Technologies (http://sg.idtdna.com/pages/home (31 July 2022)) (Table S1). The eif5a1 gene was used as the reference gene for standardizing the expressions of genes under testing [68 (link)]. The qRT-PCR analysis was performed under conditions as described previously [33 (link)]. The relative expressions of tested genes were calculated using the 2−ΔΔCt comparative Ct method. Statistical analysis of qRT-PCR results was performed using SPSS20.0, and differences with p < 0.05 were considered to be significant.
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The expression of vwa1, fgfr1a, fgfr2, fgfr3, fgfr4, fgf8a, fgf8b, and runx2a in collected embryos (n = 30) was determined by RT-qPCR using the primers shown in Supplemental Table S2. Total RNA was extracted with TRIzol (Invitrogen, Beijing, China) and purified to an A260/A280 ratio of 1.8–2.0, after which 1 μg of each sample was reverse transcribed using All-In-One 5X RT MasterMix (Abm, Beijing, China, G592). qPCR was then performed using the UltraSYBR Mixture (CWBIO, CW0957H), and expression levels of target genes were quantified using the 2−ΔΔCT method and normalized to β-actin expression levels. Expression of mRNA for vwa1-mutants is presented as fold-change relative to that of wild-type (WT) controls.
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Total RNA is extracted from uterus caruncle tissues using Trizol® reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). RNA concentration was measured by NanoDrop 2000 spectrophotometer (Thermo Fisher, Meridian, ID, USA), and the purity of RNA was determined by the ratio of OD260/280. All the ratio of OD260/280 of our RNA samples value 1.8–2.0. Each sample was reverse transcribed using All-In-One 5X RT MasterMix (Abm, Heidelberg, Germany, Cat#G592) with 2 μg total RNA.
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Total RNA was obtained from Caco-2 cells treated with or without resveratrol using TRIzol reagent (15596018, Thermo Fisher). Complementary DNA was synthesized with All-In-One 5X RT MasterMix (592, abm). Quantitative PCR was performed with a SYBR Green assay (25741, Thermo Fisher). As the gene of interest, RT-PCR was run for SIRT3, SIRT4, SIRT5, and FoxO3a. GAPDH served as a reference gene. The primer sequences were as follows: SIRT3: (forward: 5’- CCCTGGAAACTACAAGCCCAAC-3’ and reverse: 5’- GCAGAGGCAAAGGTTCCATGAG–3’); SIRT4: (forward: 5′-CAGCAAGTCCTCCTCTGGAC-3′ and reverse: 5′-CCAGCCTACGAAGTTTCTCG-3′); SIRT5: (forward: 5′-TGGCTCGGCCAAGTTCAAGTATG-3′ and reverse: 5′-AAGGTCGGAACACCACTTTCTGC-3′); FoxO3a: (forward: 5′-CTTGATGTCTCAGGCCAGCA-3′ and reverse: 5′-CAAGTCGCTGGGGAACTTCT-3′); and GAPDH: (forward: 5’- CCACCCATGGCAAATTCCATGGCA-3’, and reverse: 5’- TCTAGACGGCAGGTCAGGTCCACC-3’).
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