The largest database of trusted experimental protocols

29 protocols using EASYspin plant RNA extraction kit

With reference to the method in the kit manual, use the EASYspin plant RNA extraction kit to extract citrus RNA (Aidlab, Beijing, China). Use DS-11 spectrophotometer (DeNoVIX Inc., USA) to measure the absorbance ratio of the sample at 260 nm and 280 nm to determine the RNA concentration and purity.
Citrus total RNA was isolated using the EASYspin plant RNA extraction kit following the manufacturer’s instructions (Aidlab, Beijing, China). RNA was reverse transcribed into cDNA using the Prime ScriptRT Master Mix (TaKaRa, Ojin, Japan). RT-qPCR reaction system is 12 µL, including 6 µL the SYBRPRIME qPCR Kit (Bioround Biotechnology, Chongqing, China), 0.3 µL forward and reverse real-time primer (10 mM·L−1), 4.4 µL ddH2O and 1 µL cDNA (10 ng·µL−1). The PCR amplification conditions were: treatment at 95 °C for 2 min, then 40 amplification cycles (each at 65 °C for 10 s, 95 °C for 5 s), and finally extension at 60 °C for 15 s. Using citrus GAPDH gene (Mafra et al., 2012 (link)) as internal reference gene, the relative expression of mSDE460 in transgenic plants was calculated by the 2−ΔΔCt method (Livak and Schmittgen, 2001 (link)). The primers were listed in Supplementary Table 1. The test was repeated three times.
+ Open protocol
+ Expand
The total RNA was extracted from the leaves of both the cold-treated and control groups using an EASYspin plant RNA Extraction Kit (Aidlab China) following the manufacturer’s instructions. High-quality RNA samples (OD260/280 = 1.8 ~ 2.2, OD260/230 ≥ 2.0, RIN ≥ 6.5, 28S:18S ≥ 1.0, > 10 μg), assessed using a 2100 Bioanalyzer (Agilent) and quantified with an ND-2000 (NanoDrop Technologies), were employed to construct the sequencing library after the removal of genomic DNA using DNase I (TaKara). Each experiment included three biological replicates.
+ Open protocol
+ Expand
Total RNA was extracted from plant tissue by using the EASY spin plant RNA extraction kit (Aidlab, Beijing, China) according to the manufacturer’s instructions, including the on-column DNase treatment. RNA was quantified using a Nanodrop 1000 (Thermo Fisher Scientific) and cDNA synthesized using Hiscript Reverse Transcriptase (Vazyme, Nanjin, China) and oligo(dT) primers (Vazyme). qRT-PCR was performed using Bio-Rad SYBR Green Supermix, and PCR parameters were as follows: 95 °C for 5 min (first cycle); 40 cycles of 95 °C for 10 s, 58 °C for 10 s, and 72 °C for 30 s; and a final cycle of 72 °C for 5 min. PCRs were performed in triplicate with a Bio-Rad CFX ConnectTM Real-Time Detection System (Bio-Rad, Hercules, CA, USA). Gene expression levels were calculated by a comparative ΔΔCt method as described in the manufacturer’s instructions for the CFX ConnectTM Real-Time Detection System. All primers, including internal controls used for measurement of transcript abundance, are shown in Supplementary Table S1.
+ Open protocol
+ Expand
Total RNA was extracted using an EASYspin Plant RNA extraction kit (Aidlab, Beijing, China). TRUEscript RT MasterMix (Aidlab, Beijing, China) was used for reverse transcription using 1 μg of RNA. Primer 5 was used to design primers for quantitative real-time PCR (qRT-PCR) (Table S2). The qRT-PCR was performed using PowerUp SYBR Green Master Mix (Applied Biosystems, Carlsbad, CA, USA) in 384-well plates. An Applied Biosystems Real-Time PCR System (Life Technologies, Carlsbad, CA, USA) was used with cycling parameters: heating at 95 °C for 2 min, 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 20 s, and extension at 72 °C for 35 s. Three biological replicates were performed for each sample in order to confirm the reliability of the results. The RcGAPDH and RcACTIN genes [32 (link),33 (link)] were used as an internal quantitative control to normalize samples (Table S2). The relative expression values were calculated using the comparative CT(2−ΔΔCT) method [34 (link)]. Statistical analysis was performed using single factor ANOVA along with Least Significant Difference test using SAS 9.4, and p < 0.05 were considered significant.
+ Open protocol
+ Expand
R. chinensis ‘Old Blush’ plants were grown in temperature and light-controlled growth chamber (25 °C, 60% relative humidity, 150 µE light intensity, 16 h light/8 h darkness). The plants were cultivated in separate pots (10 × 10 × 9 cm) containing a mixture of peat moss, vermiculite and perlite (3: 2: 1) and were irrigated twice a week. For heat treatment, 3-year-old rose plants were watered once before treatment and then incubated at 35 °C for 24 h, Petals were harvested at 0, 6, 12, 24 h post-treatment. For drought treatment, the plants were irrigated once before treatment using a 200 mM mannitol solution until it dripped out from the pot bottom. Petals were harvested at 0, 12, 24, 48 h post-treatment. All samples were immediately frozen in liquid nitrogen and stored at −80 °C until use. Total RNA was extracted using EASYspin plant RNA extraction kit (AidLab, Beijing, China) and genomic DNA was removed by on-column DNAse digestion. RNA was quantified using the NanoDrop 2000c spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and RNA integrity was assessed by standard denaturing 1% TBE agarose gel electrophoresis. A total amount of 1 µg RNA was then reverse transcribed into cDNA using the TRUEscript RT MasterMix (AidLab, Beijing, China) with the following conditions: 42 °C for 20 min and 85 °C for 5 s. The cDNA was then diluted 10-fold and stored at −20 °C for qRT-PCR.
+ Open protocol
+ Expand
Total RNA from the citrus samples was isolated using the EASYspin Plant RNA Extraction Kit following the manufacturer’s instructions (Aidlab, Beijing, China). The RNA from the leaf tissues was used to determine the expression levels of CsSAMT1 in the transgenic lines. RNA was reverse transcribed into the first cDNA using the iScriptTM cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Gene expression was detected using the iQ™ SYBR Green Supermix (Bio-Rad). The PCR reactions were carried out by a pretreatment (94 °C for 5 min) followed by 40 amplification cycles (94 °C for 20 s and 60 °C for 60 s). The primers used in the qRT-PCR analysis are listed in Table S1 and all experiments were performed in triplicate. The expression of the citrus actin (GenBank No. XM_006464503.3) [13 (link),55 (link)] and GAPDH [56 (link)] genes were used for transcript normalization. Using WT plants as controls, the relative expression of CsSAMT1 in transgenic plants was calculated by the 2−ΔΔCt method [57 (link)].
+ Open protocol
+ Expand
Total RNA was extracted from floral buds using the EASYspin plant RNA Extraction Kit (Aidlab, China) following instructions from the manufacturer. First-strand cDNA was synthesized from 1 μg of the DNase I-treated RNA, using adaptor primers and M-MLV Reverse Transcriptase (TaKaRa, Japan). Initial amplification for core sequences were based on homologous cloning. The PCR reagents were composed of 1 μL cDNA, 0.5 μL of each primer (10 mM each), 2.5 μL Ex Taq buffer, 2 μL dNTP (2.5 mM each), 0.3 μL Ex Taq plymerase (TaKaRa, Japan) and adjusted with water to a final volume of 25 μL. PCR was performed with a 3 min 95°C denaturation step, followed by 35 cycles of 30 s at 95°C, 30 s annealing at 52–57°C, a 30–60 s extension at 72°C and a final extension period of 10 min. The PCR products were purified with the gel extraction kit (TaKaRa) and cloned into pMD18®-T vector (TaKaRa). Ligation products were transformed into Escherichia coli Top10 cells (Aidlab China) following instructions by the manufacturer. Then we used 3’ RACE and 5’ RACE system kits (TaKaRa) to obtain the 3’- and 5’-end sequences of each gene. Full-length cDNA of each gene was obtained by PCR-based cloning with gene-specific forward and reverse primers designed according to the corresponding 3’- and 5’-end sequences. Names and sequences of the primers used in this study are presented in Tables 1 and 2.
+ Open protocol
+ Expand
The total RNA was extracted using the EASYspin Plant RNA Extraction Kit (Aidlab, Beijing, China). After treatment with DNase I (TaKaRa, Kusatsu, Shiga, Japan), 1 µg RNA was used to synthesize the first-strand cDNA with the PrimeScript™ RT Reagent Kit (TaKaRa, Kusatsu, Shiga, Japan). Quantitative PCR was performed on a CFX96™ Real-Time System (Bio-Rad, Hercules, CA, USA) using the 1× iQ™ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) in accordance with the manufacturer’s instructions, and the data were analyzed using the native software (Bio-Rad, Hercules, CA, USA). The thermal cycling protocol consisted of a pretreatment (94 °C, 3 min) followed by 40 amplification cycles (94 °C, 30 s; 56 °C, 30 s; and 72 °C, 30 s). The data were analyzed using the CFX Maestro software installed on the CFX96™ Real-Time System (Bio-Rad, Hercules, CA, USA). Each test was confirmed by three individual runs (biological replicates), and data from one of the replicates was used to generate the expression chart. The ubiquitin gene UBQ7 (GenBank accession no. DQ116441) and actin gene ACTIN2 (AT3G18780.2) served as the reference genes in cotton and Arabidopsis, respectively. Gene-specific primers used for qRT-PCR are listed in Supplementary Materials, Table S2.
+ Open protocol
+ Expand
Total RNA was extracted using the EASY spin plant RNA extraction kit (Aidlab Biotech, China). Approximately 1 μg RNA was transcribed into first-strand cDNA using the NovoScript Plus All-in-one First Strand cDNA Synthesis SuperMix (gDNA Purge, Novoprotein, China). The real-time quantitative PCR (RT-qPCR) assays were performed on a CFX Connect Real-Time System (Bio-Rad Laboratories) with 2×NovoStart SYBR qPCR SuperMix plus (Novoprotein, China). GhHis3 (Zhang et al., 2011 ; Wan et al., 2016 (link); Wu et al., 2018 (link)) and GhUbiquitin (Walford et al., 2011 (link); Wu et al., 2018 (link)) served as internal references. Gene specific primers used for RT-qPCR are listed in Supplementary Table S2. The expression data were calculated with the ΔΔCt method. For the RT-qPCR analysis, three individual biological replicates with two technical replicates for each gene were used. Mean values and standard errors were calculated using the data from the three replicates. The compliance of the RT-qPCR experiments with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) is shown in a MIQE checklist (Supplementary Table S3).
+ Open protocol
+ Expand
Total RNA was isolated using the EASYspin Plant RNA extraction kit (Aidlab, Beijing, China) following the manufacturer’s protocol. All libraries (three biological replicates for every sample) were sequenced on the BGI platform. Transcriptome data for nine samples of different petal stages of ‘OB’ were downloaded from NCBI (PRJNA351281) (Han et al. 2017 (link)). After quality control and the removal of low-quality and adapter-containing reads with Trimmomatic–0.36, the clean data were aligned to the hB genome of ‘CH’ and ‘OB’ using HiSAT2 v2.1.0 (Kim et al. 2015 (link)). For accurate quantification of homologous gene expression, only the unique mapping reads were kept for further analysis. Transcript levels were normalized using the fragments per kilobase per million mapped reads (FPKM) method and coverages of exon were calculated with StringTie v1.3.5 (Pertea et al. 2016 (link)).
Differential expression analysis of two conditions/groups was performed using the DESeq2 (Love et al. 2014 (link)). The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted P-value < 0.05 (log2FoldChange > 2 | log2FoldChange < -2) found by DESeq2 were assigned as differentially expressed. The Venn diagram and kyoto encyclopedia of genes and genomes (KEGG) enrichment diagram were performed using R software.
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!