Total RNA was treated with DNase I (Takara, Japan) to remove residual genomic DNA. For the qRT-PCR analysis of AtMYB33 and MYB65, AtACTIN2 gene was used as internal control. PrimeScript™ II reverse transcriptase (Takara) and oligo (dT) primers were used for first-strand cDNA synthesis. For the qRT-PCR analysis of mature miRNAs, U6 was used as internal control gene, and the first cDNA was synthetized using a miRNA First Strand cDNA Synthesis kit (Stem-loop Method) (Sangon Biotech). The qRT-PCR reactions were performed in a MyiQ2 qRT-PCR detection system (Bio-Rad, www.bio-rad.com/ ) using iQ SYBR Green supermix (Bio-Rad) [42 (link)]. Each experiment was conducted in three biological replicates, and the same sample was performed in three technical replicates. Relative expression levels of miRNAs and their target genes were quantified by using the 2-ΔΔCt method [43 (link)]. All the primers used for qRT-PCR analysis are listed in Additional file 1 : Table S4.
Mirna first strand cdna synthesis kit stem loop method
Manufactured by Sangon
Sourced in China
The MiRNA First Strand cDNA Synthesis kit (Stem-loop Method) is a product designed for the reverse transcription of mature microRNA (miRNA) molecules. The kit utilizes a stem-loop primer method to selectively convert mature miRNA into complementary DNA (cDNA) for downstream applications such as real-time PCR analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Rneasy minelute cleanup kit, by Qiagen (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Primescript rt master mix, by Takara Bio (2 mentions)
Tg green premix ex taq 2 kit, by Takara Bio (2 mentions)
Rnase r, by Illumina (2 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Micrornas qpcr kit sybr green method, by Sangon (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Stratagene mx3000p system, by Agilent Technologies (2 mentions)
Microrna qrt pcr kit sybr green method, by Sangon (2 mentions)
Dnase 1, by Takara Bio (1 mentions)
Primescript 2 reverse transcriptase, by Takara Bio (1 mentions)
Iq sybr green supermix, by Bio-Rad (1 mentions)
Myiq2 qrt pcr detection system, by Bio-Rad (1 mentions)
Evagreen 2x qrt pcr mastermix low rox, by Applied Biological Materials (1 mentions)
Evagreen 2 qrt pcr mastermix low rox, by Applied Biological Materials (1 mentions)
5 protocols using mirna first strand cdna synthesis kit stem loop method
1
RT-qPCR Analysis of miRNAs and Target Genes
2
RNA Extraction, Fractionation, and Expression Analysis
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RNA was extracted using TRIzol reagent (Invitrogen, CA, USA). For RNase R treatment, total RNA (2 μg) was incubated with or without RNase R (3 U/μg; Epicentre Technologies, Madison, WI, USA) in for 20 min at 37 °C, and the resulting RNA was purified using an RNeasy MinElute cleanup Kit (Qiagen). For cellular RNA fractionation analysis, the cells' nuclear and cytoplasmic fractions were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific).
For circBCBM1 and BRD4 expression detection, the cDNAs were synthesized using PrimeScript RT Master Mix (Takara, Dalian, China) according to the manufacturer's instructions. The RT-qPCR was performed using TG Green Premix Ex Taq II kit (Takara, Dalian, China) with a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) as previously described 19 (link). GAPDH served as an internal reference gene. For miR-125a expression detection, the cDNAs were synthesized using miRNA First Strand cDNA Synthesis kit (Stem-loop Method; Shanghai Sangon Biotech, Shanghai, China) according to the manufacturer's instructions. Then qPCR was performed using MicroRNAs qPCR Kit (SYBR Green Method; Shanghai Sangon Biotech, Shanghai, China). U6 served as an internal reference gene. All primer sequences are listed in SupplementaryTable S1 .
For circBCBM1 and BRD4 expression detection, the cDNAs were synthesized using PrimeScript RT Master Mix (Takara, Dalian, China) according to the manufacturer's instructions. The RT-qPCR was performed using TG Green Premix Ex Taq II kit (Takara, Dalian, China) with a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) as previously described 19 (link). GAPDH served as an internal reference gene. For miR-125a expression detection, the cDNAs were synthesized using miRNA First Strand cDNA Synthesis kit (Stem-loop Method; Shanghai Sangon Biotech, Shanghai, China) according to the manufacturer's instructions. Then qPCR was performed using MicroRNAs qPCR Kit (SYBR Green Method; Shanghai Sangon Biotech, Shanghai, China). U6 served as an internal reference gene. All primer sequences are listed in Supplementary
3
Circular RNA and mRNA Detection
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For RNase R treatment, total RNA (2 μg) was incubated with or without RNase R (3 U/μg; Epicentre Technologies, Madison, WI, USA) in 1×RNase R reaction buffer for 20 min at 37 °C, and the resulting RNA was puri ed using an RNeasy MinElute cleanup Kit (Qiagen). For cellular RNA fractionation analysis, the cells' nuclear and cytoplasmic fractions were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scienti c). The total RNA was extracted using TRIzol reagent (Invitrogen, CA, USA).
For circBCBM1 and BRD4 detection, the cDNAs were synthesized using PrimeScript RT Master Mix (Takara, Dalian, China) according to the manufacturer's instructions. The RT-qPCR was performed using TG Green Premix Ex Taq II kit (Takara, Dalian, China) with a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) as previously described [18] . GAPDH served as an internal reference gene. For miR-125a detection, the cDNAs were synthesized using miRNA First Strand cDNA Synthesis kit (Stemloop Method; Shanghai Sangon Biotech, Shanghai, China) according to the manufacturer's instructions.
Then qPCR was performed using MicroRNAs qPCR Kit (SYBR Green Method; Shanghai Sangon Biotech, Shanghai, China). U6 served as an internal reference gene. All primer sequences are listed in Supplementary Table S1.
For circBCBM1 and BRD4 detection, the cDNAs were synthesized using PrimeScript RT Master Mix (Takara, Dalian, China) according to the manufacturer's instructions. The RT-qPCR was performed using TG Green Premix Ex Taq II kit (Takara, Dalian, China) with a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) as previously described [18] . GAPDH served as an internal reference gene. For miR-125a detection, the cDNAs were synthesized using miRNA First Strand cDNA Synthesis kit (Stemloop Method; Shanghai Sangon Biotech, Shanghai, China) according to the manufacturer's instructions.
Then qPCR was performed using MicroRNAs qPCR Kit (SYBR Green Method; Shanghai Sangon Biotech, Shanghai, China). U6 served as an internal reference gene. All primer sequences are listed in Supplementary Table S1.
4
Quantitative Analysis of RNA Transcripts
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Total RNA was isolated from cells and tissues using TRIzol reagent (Invitrogen, US) according to the manufacturer’s procedural guidelines. For mRNA and lncRNA quantification, the RNA was reverse transcribed into cDNA with a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, US). EvaGreen 2X qRT-PCR MasterMix-Low ROX (abm, Canada) was used for quantitation with specific primers for the mRNA and lncRNA. GAPDH was used as an internal control. For miRNA quantification, reverse transcription was performed using an miRNA First Strand cDNA Synthesis (Stem-loop Method) Kit (Sangon Biotech, Shanghai, China). A MicroRNA qRT-PCR Kit (SYBR Green Method) (Sangon Biotech, Shanghai, China) was used for quantitation with specific primers for miRNA. U6 was used as an internal control. The primer sequences are listed in Additional file 2 : Table S1. All quantitative real-time PCR experiments were performed with an Agilent Technologies Stratagene Mx3000P system (Agilent Technologies, US). The data were processed with the 2-ΔΔCt method, and the fold changes were normalized to the expression of the internal controls.
5
Quantitative Analysis of RNA Expression
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Total RNA was isolated from cells using TRIzol reagent (Invitrogen, USA). For mRNA and lncRNA quantification, the RNA was reverse transcribed into cDNA with a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, USA). EvaGreen 2× qRT-PCR MasterMix-Low ROX (abm, Canada) was used for quantitation with specific primers for the mRNA and lncRNA. GAPDH was used as an internal control. For miRNA quantification, reverse transcription was performed using an miRNA First Strand cDNA Synthesis (Stem-loop Method) Kit (Sangon Biotech, Shanghai, China). A MicroRNA qRT-PCR Kit (SYBR Green Method) (Sangon Biotech, Shanghai, China) was used for quantitation with specific primers for miRNA, and U6 was used as an internal control. The primer sequences were listed in Table S1 . All quantitative real-time PCR experiments were performed with an Agilent Technologies Stratagene Mx3000P system (Agilent Technologies, USA). The data was processed with the 2-ΔΔCt method, and the fold changes were normalized to the expression of the internal controls.
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