To explore the function of the MeGLYI-13 gene in iron stress, 40-day-old SC8 cassava seedlings were treated with 450 μmol/L of FeCl3 to induce excessive iron stress. Meanwhile, 40-day-old SC8 cassava seedlings without treatment were used as the control. Root, shoot, and leaf samples were harvested at 0, 2, 6, 12, and 24 h after the iron treatment, separately. In this experiment, two plants and three biological replicates were set for each treatment. Then, all of the samples were frozen by liquid nitrogen for isolating the total RNA using a Plant Total RNA Isolation Kit Plus (Foregene). The MonScript™ RTIII Super Mix with dsDNase (Two-Step) Kit was used to remove the remaining DNA from the RNA and then for reverse transcription of the RNA into cDNA. The qRT-PCR data were reacted by SYBR® Premix Ex TaqTM II reagent (Takara) and detected by the ABI 7900 HT Fast Real-Time PCR System. The relative expression levels of MeGLYI-13 at different times after treatment were calculated and analyzed by the popular 2−ΔΔCT method. The primers used in this experiment are listed in Table S3 .
Plant total rna isolation kit plus
Manufactured by Foregene
Sourced in China
The Plant Total RNA Isolation Kit Plus is a laboratory equipment designed to efficiently extract and purify total RNA from various plant species. It provides a reliable and consistent method for isolating high-quality RNA suitable for downstream applications such as RT-PCR, northern blotting, and RNA-seq.
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Lab products found in correlation
Primescript rt reagent kit with gdna eraser, by Takara Bio (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Lightcycler sybr green 1 master mix kit, by Roche (3 mentions)
Primescript rt reagent kit, by Takara Bio (3 mentions)
Lightcycler 480 instrument 2, by Roche (2 mentions)
Transscript one step gdna removal and cdna synthesis supermix kit, by Transgene (2 mentions)
Primescript 2 1st strand cdna synthesis kit, by Takara Bio (2 mentions)
Lightcycler 480 2, by Roche (2 mentions)
Sybr premix ex taqtm 2 reagent, by Takara Bio (1 mentions)
Abi 7900 ht fast real time pcr system, by Takara Bio (1 mentions)
Transscript one step gdna removal and cdna synthesis supermix, by Transgene (1 mentions)
Primerscript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Tb green premix ex taq, by Takara Bio (1 mentions)
Transstart tip green qpcr supermix kit, by Transgene (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Quant gene 9600 system, by Bioer (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Magicsybr mixture, by CWBIO (1 mentions)
Minute chloroplast isolation kit, by Invent Biotechnologies (1 mentions)
Lightcycler 480 sybr green 1 master, by Roche (1 mentions)
21 protocols using plant total rna isolation kit plus
1
Investigating MeGLYI-13 Gene's Role in Cassava Iron Stress
2
Pear Fruit RNA Extraction Protocol
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We collected six pear accessions including three wild accessions of P. pyrifolia, ‘Matanggengzi’ (‘MTGZ’), ‘Baitanggengzi’ (‘BTGZ’), and ‘Tiantanggengzi’ (‘TTGZ’), and three cultivated accessions, ‘Huanghuali’ (‘HH’), ‘Lipuxueli’ (‘LPXL’), and ‘Liuchengfengshan’ (‘LCFS’), at three fruit developmental stages (small fruit stage, 52 DAF; enlarged fruit stage, 94 DAF; mature fruit stage, 128 DAF) for qRT-PCR analysis. First, we mixed the pear fruit samples of the same developmental stages from wild or cultivated genotypes. Then, the Plant Total RNA Isolation Kit Plus (FOREGENE Co. Ltd.) was used to extract the total RNA from the mixed samples of pear fruit. In the process, we carried out an improved step that was proposed in our previous study [31 (link)]. To obtain higher-quality RNA from pear fruit at the late stage of development, with high water content, less water (40 μl) was used to elute the RNA from the filtration column. Then, the total RNA was adjusted to the same concentration, and based on the adjusted RNA, first-strand cDNA was synthesized using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech Co. Ltd.).
3
Total RNA Extraction and Purification
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Total RNA was extracted from the leaf samples using a Plant Total RNA Isolation Kit Plus (Foregene, Chengdu, China) according to the manufacturer's instructions. RNA integrity was checked by 1% agarose gel electrophoresis, and RNA concentration and purity were measured using a Nanodrop ND 1000 Spectrophotometer. Samples with 28S/18S ribosomal RNA between 1.5 and 2.0 and an absorbance ratio OD260/280 between 1.9 and 2.1 were used in the subsequent transcriptomic analysis. Approximately 1,000 ng of total RNA was used for cDNA synthesis using a PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). The cDNA was diluted with nuclease-free water prior to conducting RT-qPCR.
4
Quantitative RT-PCR Analysis of PbrPUBs
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Total RNA of leaves materials under stress was extracted using Plant Total RNA Isolation Kit Plus (FOREGENE, China). Then, PrimeScript™ RT reagent kit (Takara Bio, China) was used to reverse transcribe RNA to cDNA. QRT-PCR analysis was conducted on Roche LightCycler® 480 II (Roche, Mannheim, Germany) using LightCycler® SYBR GREEN I Master Mix kit (Roche, China). We designed fifteen pairs specific primers (Additional file 4 : Table S1) using Primer5.0 software and checked by using NCBI online software (https://www.ncbi.nlm.nih.gov/ ) . The reaction system and protocol of qRT-PCR were consistent with our previously study [59 (link), 62 (link)]. The relatively expression level of PbrPUBs were estimated using 2−∆∆CT method [63 (link)]. The pear Tubulin gene (No. AB239681) was selected as an internal reference for Pyrus betulaefolia, and the Actin gene (No. AY063980) was selected as an internal reference for Arabidopsis.
5
Drought and Salt Stress Response in Woodland Strawberry
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Woodland strawberry plants (Fragaria vesca, Hawaii4) were grown in greenhouse at Nanjing Agricultural University (Jiangsu, China), and maintained at room temperature under a photoperiod of 16/8 h. Two-month-old plants were used for drought and salt treatment. Tender (the stage before fully expanded mature leaves) and old (the stage of fully expanded mature leaves) leaf samples were collected at 0, 3, 5, and 7 days after water was withheld. In addition, strawberry leaves were used as salt stress treatment and samples were collected at 0, 3, 5, and 7 days after treatment.
Total RNA was extracted using the Plant Total RNA Isolation Kit Plus (Foregene, Chengdu, China). First strand cDNA was synthesized using PrimerScript™ RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). RT-qPCR analysis was performed by Light Cycler 480 II (Roche, Switzerland) with TB Green® Premix Ex Taq™ (TaKaRa, Dalian, China). 18sRNA was used as an internal reference gene to normalize the expression of GRAS genes using the 2−△△Ct method [59 (link)]. Each set of RT-qPCR data was calculated with three replicates. The primer sequences used for RT-qPCR were listed in Table S5 .
Total RNA was extracted using the Plant Total RNA Isolation Kit Plus (Foregene, Chengdu, China). First strand cDNA was synthesized using PrimerScript™ RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). RT-qPCR analysis was performed by Light Cycler 480 II (Roche, Switzerland) with TB Green® Premix Ex Taq™ (TaKaRa, Dalian, China). 18sRNA was used as an internal reference gene to normalize the expression of GRAS genes using the 2−△△Ct method [59 (link)]. Each set of RT-qPCR data was calculated with three replicates. The primer sequences used for RT-qPCR were listed in Table S
6
Sesame Gene Expression Analysis
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The RNA extraction from three tissues (root, stem, and leaves) of control and treatment was performed using the Plant Total RNA Isolation Kit Plus (FOREGENE, China). Subsequently, the PrimeScript™ RT reagent kit (Takara Bio, China) was utilized to reverse transcribe the extracted RNA into cDNA. For qRT-PCR analysis, the Roche LightCycler® 480 II instrument (Roche, Mannheim, Germany) was employed with LightCycler® SYBR GREEN I Master Mix kit (Roche, China). We designed 7 pairs of specific primers (Supplementary Table S1
7
Quantification of GhBASSs Expression
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Total RNA was extracted using the Plant Total RNA Isolation Kit Plus (Foregene, Chengdu, China) with the manufacturer’s guidelines. The TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen Biotech, Beijing, China) was used for the first-strand cDNA synthesis following the supplier’s protocols. The relative quantification of GhBASSs by qPCR was conducted with SYBR Green on a LightCycler 480 (Roche Molecular Systems, Inc., Indianapolis, Indiana, USA). Following the instructions of the TransStart Tip Green qPCR SuperMix kit (TransGen Biotech, Beijing, China), the reaction mixture and PCR cycles were performed. The cotton UBQ 7 (GenBank accession no. DQ116441) and Arabidopsis UBQ 10 (GenBank accession no. AT4G05320) were applied as internal references for normalization. The transcript levels of GhBASSs were calculated by the comparative 2−ΔΔCT method81 (link), and three independent biological replicates were done for each assay. Primers used for qPCR analyses were listed in Table S3 .
8
Quantitative PCR of Cotton Leaf RNA
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According to the manufacturer’s instructions, the total RNA from cotton leaves was extracted using the Plant Total RNA Isolation Kit Plus (FOREGENE, Chengdu, China). The quality and concentration of the RNA samples were assessed using 1% agarose gel electrophoresis, and a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). The extracted RNA was reverse transcribed into cDNA using the FOREGENE (Chengdu, China) Master Premix (For qPCR) RT EasyTM II reverse transcription kit. Real-time fluorescence quantitative PCR was performed using the Quant Gene 9600 system (Bioer Technology, Hangzhou, China) with the following reaction conditions: 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s, 58 °C for 10 s, 72 °C for 20 s, 95 °C for 15 s, 58 °C for 1 min, and 95 °C for 15 s. The GhHis3 gene of cotton was used as an internal reference gene, and the expression level of the target gene was calculated using the 2−ΔΔCt method. All analyses were performed in triplicate biological and technical replicates. The primers used in this study are listed in Supplementary Table S3 .
9
Total RNA Extraction and cDNA Synthesis
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Total RNA was extracted using Plant Total RNA Isolation Kit Plus (FOREGENE, Chengdu, China) according to the manufacturer’s instructions. The concentration and purity of extracted RNA was measured by a NanoDrop2000 (Thermo Scientific, USA), and its integrity was evaluated by 1.8% (w/v) agarose gel electrophoresis. Only the RNA absorbing ratio of 1.8–2.0 at OD260 nm/OD280 nm were used for further cDNA synthesis with the Prime Script™ RT reagent Kit with gDNA Eraser (TaKaRa, RR047A). The synthesized cDNAs were verified by RT-PCR and diluted tenfold for qRT-PCR analyses.
10
RNA Extraction and Preparation for RT-qPCR
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Frozen samples were ground to a fine powder in liquid nitrogen and total RNA was extracted using Plant Total RNA Isolation Kit Plus (Foregene, Chengdu, China). RNA integrity was checked by 1% agarose gel electrophoresis and RNA quality was measured by Nanodrop ND 1000 spectrophotometer (Nanodrop Technologies Inc, Delaware, USA). Samples with 28S/18S ribosomal RNA between 1.5 and 2.0 and an absorbance ratio OD260/280 between 1.9 and 2.2 were used for subsequent experiments. Approximately 1,000 ng total RNA was used for cDNA synthesis using the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). The cDNA was diluted with nuclease-free water before RT-qPCR.
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