Total RNA was extracted from the chick cerebellum using Sepazol-RNA I Super (Nacalai Tesque, Kyoto, Japan) and reverse transcribed as described previously (Haraguchi et al., 2010 (link); Haraguchi et al., 2012a (link); Haraguchi et al., 2012b (link); Haraguchi et al., 2015 (link); Nozaki et al., 2018 (link)). All PCR amplifications (for mPRα, mPRβ, mPRγ, PXR, and α1-subunit of the GABAA receptor) were performed in a reaction mixture containing Ex Taq polymerase (Takara, Shiga, Japan). Forward and reverse primers (Table 1 ) were designed according to the nucleotide sequence of chicken mRNAs. The following PCR conditions were used for the thermal cycler: 1 cycle of 1 min at 94°C, 30 cycles of 30 s at 94°C, 30 s at 60°C, 30 s at 72°C, and finally, 1 cycle of 10 min at 72°C. The identities of the PCR products were confirmed by sequencing.
Sepazol rna 1 super
Manufactured by Nacalai Tesque
Sourced in Japan
Sepazol-RNA I Super is a reagent designed for the extraction and purification of total RNA from various biological samples. It is a monophasic solution containing phenol and guanidinium thiocyanate, which facilitates the lysis of cells and the inactivation of RNases. The product allows for the efficient and reliable isolation of high-quality RNA suitable for downstream applications such as RT-PCR, Northern blotting, and gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ex taq polymerase, by Takara Bio (1 mentions)
Ptc 100, by Bio-Rad (1 mentions)
Rq1 dnase stop solution, by Promega (1 mentions)
Rnase inhibitor, by Toyobo (1 mentions)
Reverse transcription buffer, by Toyobo (1 mentions)
Oligo dt 20, by Toyobo (1 mentions)
Deoxyribonucleotide triphosphate dntp mixture, by Toyobo (1 mentions)
Nanodrop lite, by Thermo Fisher Scientific (1 mentions)
Revertra ace, by Toyobo (1 mentions)
Rq1 rnase free dnase, by Promega (1 mentions)
2 protocols using sepazol rna 1 super
1
RNA Extraction and RT-PCR of Chick Cerebellum
2
Ileum and Cecum RNA Extraction, DNase Treatment, and Reverse Transcription
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RNA samples were extracted from the ileum and cecum using Sepazol RNA I Super (Nacalai Tesque Inc., Kyoto, Japan) according to the manufacturer's directions. RNA was dissolved in 10 mM Tris with 1 mM EDTA (pH 8.0) and stored at −80°C until use.
The RNA was treated with 1U RQ1 RNase-free DNase (Promega Co, Madison, WI, USA) in a 10-µl reaction mixture (10 µg total RNA, 1× DNase buffer and 1 U DNase) on a programmable thermal controller (PTC-100; MJ Research, Waltham, MA, USA) programmed at 37°C for 30 min and then at 65°C for 10 min. The reaction was stopped with 1U RQ1 DNase Stop Solution (Promega Corporation, Madison, USA). The concentration of RNA was measured using a NanoDrop Lite instrument (Thermo Fisher Scientific, Waltham, WV, USA). The RNA was then reverse-transcribed using ReverTra Ace (Toyobo Co. Ltd., Osaka, Japan) according to the manufacturer's instructions. The reaction mixture (10 µl) consisted of 0.5 µg total RNA, 1× reverse transcription buffer (Toyobo Co. Ltd.), 1 µM deoxyribonucleotide triphosphate (dNTP) mixture (Toyobo Co. Ltd.), 5 U RNase inhibitor (Toyobo Co. Ltd.), 0.25 µg of oligo(dT)20 (Toyobo Co. Ltd.), and 50 U ReverTra Ace. Reverse transcription was carried out at 42°C for 30 min, followed by heat inactivation at 99°C for 5 min, in a programmable thermal controller (PTC-100; MJ Research). The cDNA samples were stored at −20°C until use.
The RNA was treated with 1U RQ1 RNase-free DNase (Promega Co, Madison, WI, USA) in a 10-µl reaction mixture (10 µg total RNA, 1× DNase buffer and 1 U DNase) on a programmable thermal controller (PTC-100; MJ Research, Waltham, MA, USA) programmed at 37°C for 30 min and then at 65°C for 10 min. The reaction was stopped with 1U RQ1 DNase Stop Solution (Promega Corporation, Madison, USA). The concentration of RNA was measured using a NanoDrop Lite instrument (Thermo Fisher Scientific, Waltham, WV, USA). The RNA was then reverse-transcribed using ReverTra Ace (Toyobo Co. Ltd., Osaka, Japan) according to the manufacturer's instructions. The reaction mixture (10 µl) consisted of 0.5 µg total RNA, 1× reverse transcription buffer (Toyobo Co. Ltd.), 1 µM deoxyribonucleotide triphosphate (dNTP) mixture (Toyobo Co. Ltd.), 5 U RNase inhibitor (Toyobo Co. Ltd.), 0.25 µg of oligo(dT)20 (Toyobo Co. Ltd.), and 50 U ReverTra Ace. Reverse transcription was carried out at 42°C for 30 min, followed by heat inactivation at 99°C for 5 min, in a programmable thermal controller (PTC-100; MJ Research). The cDNA samples were stored at −20°C until use.
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