Total RNA was extracted from the cells using a Total RNA Extraction Kit (Generay Biotech, Shanghai, China), RNA was reverse-transcribed into cDNA using a RevertAid First Strand cDNA synthesis Kit (Thermo Scientific Fisher, Waltham, MA, United States), and the thermocycling program used was as follows: 37°C for 60 min and 85°C for 5 min. Amplifications were performed in an iCycler using iQ SYBR Green Supermix (Bio-Rad). GAPDH was amplified on the same plates and used to normalize the data. Each sample was prepared in triplicate, and each experiment was repeated at least three times. The relative abundance of each gene was quantified using the 2–ΔΔCt method. The PCR primers used are listed in Supplementary Table S1 .
Total rna extraction kit
Manufactured by Generay
Sourced in China
The Total RNA Extraction Kit is a laboratory tool designed to isolate and purify total RNA from a variety of biological samples. The kit utilizes a guanidinium-based lysis and column-based purification method to efficiently extract high-quality RNA for downstream analysis.
Automatically generated - may contain errors
Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Iq sybr green supermix, by Bio-Rad (2 mentions)
Qubit rna assay kit in qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Rna nano 6000 assay kit, by Agilent Technologies (1 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Nanophotometer, by Implen (1 mentions)
Bulge loop mirna qrt pcr primer set, by RiboBio (1 mentions)
Bulge loop mirna qrt pcr starter kit, by RiboBio (1 mentions)
U6 snrna qpcr primer set, by RiboBio (1 mentions)
3 protocols using total rna extraction kit
1
Real-Time qRT-PCR for Gene Expression
2
Transcriptome Analysis of Plant Responses
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Total RNA free of genomic DNA was extracted using a Total RNA Extraction Kit (Generay, China), following the provided protocols. Purity and quality were assessed by 1% agarose gel electrophoresis and spectrophotometry (Nanophotometer, IMPLEN, CA, USA) using the A260/A280 and A260/A230 ratios. RNA concentrations were determined with a Qubit® RNA Assay Kit in Qubit®2.0 Fluorometer (Life Technologies, CA, USA) and integrity was measured with the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). We then mixed equal amounts of RNA samples from leaves and tuberous roots at each time point under heat, drought, and their co-stresses to construct four sequencing libraries, namely, HT, DR, DH, and CK (control). Each library was constructed and analyzed in triplicate resulting in 12 sequencing libraries for 12 pooled samples.
3
Quantification of miRNA and mRNA Expression
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Total RNA was extracted from the cells using a Total RNA Extraction Kit (Generay Biotech, Shanghai, China), then RNA was reverse-transcribed into cDNA using a RevertAid First Strand cDNA synthesis Kit (Thermo Scientific Fisher, Waltham, MA, UK) and the thermocycling program used was as follows: 37℃ for 60 mins and 85℃ for 5mins. Amplifications were performed in an iCycler using iQ SYBR Green supermix (Bio-Rad). Gapdh was amplified on the same plates and used to normalize the data. Each sample was prepared in triplicate and each experiment was repeated at least three times. The relative abundance of each gene was quantified using the 2 -ΔΔCt method. The PCR primers used are listed in Table S1. Total RNA for miR-3605-5p, miR-6082-3p, and U6 detection was extracted with a Total RNA Extraction Kit. Reverse transcription and PCR was performed using a BulgeLoopTM miRNA qRT-PCR Starter Kit, a Bulge-Loop™ miRNA qRT-PCR Primer Set, and a U6 snRNA qPCR Primer Set (RiboBio, Guangzhou, China) according to the manufacturer's instructions. miRNA expression was quantified using the 2 -ΔΔCt method and U6 was used as an internal control.
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