Total RNA was extracted with a modified CTAB method [4 (link)]. cDNA library was generated by Primerscript RT reagent Kit with gDNA Erase (Takara, Beijing, China) according to the manufacturer’s protocol. qPCR was carried out using SYBR Premix Ex Taq II (Takara, Beijing, China). Primer sequences were listed in Table S6 . Three biologicals with triplicates were performed and results were analyzed using the 2−ΔCT method. Actin gene (gene ID: Solyc11g005330) was used as the reference.
Primerscript rt reagent kit with gdna erase
Manufactured by Takara Bio
Sourced in China
The Primerscript RT reagent Kit with gDNA Erase is a product offered by Takara Bio. It is designed for reverse transcription of RNA. The kit includes reagents required for the reverse transcription process.
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Lab products found in correlation
4 protocols using primerscript rt reagent kit with gdna erase
1
Total RNA Extraction and qPCR Analysis
2
Quantitative Gene Expression Analysis in Strawberry
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Before RNA extraction, the achenes were stripped from the frozen strawberry fruit, and only the de-seeded flesh was processed for RNA isolation. RNA was isolated from either the deseeded flesh or seedlings by a modified CTAB method. After DNase I treatment, RNAs were used for cDNA synthesis by using the Primerscript RT reagent Kit with gDNA Erase (Takara). The cDNAs were used as templates for quantitative RT-PCR to measure the abundance of a certain transcript. Quantitative RT-PCR was performed using SYBR Premix Ex Tag (Takara) on a Bio-rad iQ5. Primers used are listed in Supplementary Table S6 . Results were analyzed by using the ΔΔCT method42 (link) using GAPDH as the control locus43 (link). Three biological and three technical replicates were performed and analyzed.
3
RT-qPCR Analysis of Gene Expression
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Total RNA of each sample was extracted with a modified CTAB method [67 (link)]. Primerscript RT reagent Kit with gDNA Erase (Takara) was used to generate cDNA according to the manufacturer’s protocol. RT-qPCR reactions were carried out with SYBR Premix Ex Taq II (Takara) on a Bio-Rad CFX96. The relative expression levels were analyzed with the ΔΔCT method using FveCHC1 as the reference gene [68 (link)]. Three biological and three technical replicates were performed for RT-qPCR. All of the specific primers we used for RT-qPCR were listed in Table S5 . The specificity and amplification efficiency of the RT-qPCR primers were validated through PCR amplification and the melting curves generated by the RT-qPCR system during the reactions.
4
Tissue Sampling and RNA Extraction Protocol for Adult Fish
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Various tissues of adult fish were dissected according to the method as previously described (Tripti and Mary, 2010 (link)). Tissues were rapidly frozen in liquid nitrogen and stored at −80°C until use. Total RNA for cloning and quantitative reverse transcription polymerase chain reaction (qRT-PCR) was extracted from fish brain, liver, gill, ovary, muscle, skin, heart, gut, kidney, and spleen using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. RNA quality was assessed by electrophoresis on a 1% agarose gel, and RNA concentration was estimated using a Nanodrop 2000 spectrophotometer (Thermo Fisher).
One microgram of total RNA was reverse transcribed using PrimerScript™ RT reagent Kit with gDNA Erase (Takara). qRT-PCR was carried out with FastStart SYBR Green Master (Roche), and each reaction was triplicated to avoid possible random variations. Primers were designed according to various hepcidin sequences obtained from the National Center for Biotechnology Information (NCBI). Primers are shown inTable 1 .
One microgram of total RNA was reverse transcribed using PrimerScript™ RT reagent Kit with gDNA Erase (Takara). qRT-PCR was carried out with FastStart SYBR Green Master (Roche), and each reaction was triplicated to avoid possible random variations. Primers were designed according to various hepcidin sequences obtained from the National Center for Biotechnology Information (NCBI). Primers are shown in
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