Total RNA was extracted from the CC using RNAiso plus reagent (Takara Bio Inc.), according to the manufacturer's recommended protocol. The RNA concentration and quality were measured via spectrophotometry at 260 and 280 nm using a Nanodrop 2000 (Thermo Fisher Scientific Inc.). Then, 1 μg of total RNA was reverse transcribed using ReverTra Ace (Toyobo Co. Ltd), dNTP mixture (Toyobo), RNase inhibitor (Toyobo), and oligo (dT)20 (Toyobo) in a TaKaRa PCR Thermal Cycler PERSONAL (Takara Bio). cDNA samples were diluted five times with RNase‐free water. Real‐time PCR was performed with KAPA SYBR green (KAPA Biosystems Inc.) according to the manufacturer's recommended protocol. All primer sequences are shown in Table 1 . Initial denaturation was performed at 95℃ for 3 min using a CFX Connect Real‐Time PCR Detection System (Bio‐Rad Laboratories Inc.). Then, amplification was performed for 40 cycles of 95℃ for 3 s and 60℃ for 30 s. The specificity of each primer was confirmed by melt curve analysis. The data were analyzed using the ΔΔCt method, which indicates relative changes in gene expression normalized to the housekeeping gene, β‐actin.
Oligo dt 20
Manufactured by Toyobo
Sourced in Japan
Oligo(dT)20 is a synthetic oligonucleotide composed of 20 deoxythymidine (dT) residues. It is commonly used in molecular biology applications as a primer for the reverse transcription of polyadenylated (poly(A)) RNA to produce complementary DNA (cDNA).
Automatically generated - may contain errors
Lab products found in correlation
Revertra ace, by Toyobo (7 mentions)
Rnase inhibitor, by Toyobo (4 mentions)
Ptc 100, by Bio-Rad (4 mentions)
Reverse transcription buffer, by Toyobo (4 mentions)
Nanodrop lite, by Thermo Fisher Scientific (4 mentions)
Deoxyribonucleotide triphosphate dntp mixture, by Toyobo (4 mentions)
Sepasol rna 1 super, by Nacalai Tesque (2 mentions)
Rq1 rnase free dnase mixture, by Promega (2 mentions)
1urq1 dnase stop solution, by Promega (2 mentions)
Rq1 dnase stop solution, by Promega (2 mentions)
Rq1 rnase free dnase, by Promega (2 mentions)
Takara pcr thermal cycler personal, by Takara Bio (1 mentions)
Kapa sybrgreen, by Roche (1 mentions)
Rnaiso plus reagent, by Takara Bio (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (1 mentions)
Dntp mixture, by Toyobo (1 mentions)
Sepasol rna 1 super g, by Nacalai Tesque (1 mentions)
Luna universal qpcr master mix, by New England Biolabs (1 mentions)
First strand cdna synthesis kit, by Toyobo (1 mentions)
8 protocols using oligo dt 20
1
Quantitative RT-PCR Gene Expression Analysis
2
Real-time qPCR for BIRC5 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed with PBS and then cultured in RPMI medium containing 1% FBS or 10% FBS without IL-3, or treated with CPT or FL118 in the same mediums for indicated periods. These cells were treated with imatinib. Total RNA was isolated from K562 cells, transduced K562 cells, and transduced Ba/F3 cells using Sepasol-RNA I Super G (Nacalai Tesque) according to the manufacturer’s instructions. cDNAs were synthesized using ReverTra Ace and oligo dT20 (TOYOBO, Osaka, Japan). Real-time PCR using Luna Universal qPCR Master Mix (NEW ENGLAND Biolabs, MA, USA) was performed as previously described [54 (link)]. PCR primer sequences were as follows: human BIRC5; (forward) 5′-TTCTCAAGGACCACCGCATC-3′ and (reverse) 5′-AAGACATTGCTAAGGGGCCC-3′, human β2 microglobulin; (forward) 5′-CTCACGTCATCCAGCAGAGA-3′ and (reverse) 5′-CGGCAGGCATACTCATCTTT-3′, mouse birc5; (forward) 5′-CCAGATCTGGCAGCTGTACC-3′ and (reverse) 5′-TGGCTCTCTGTCTGTCCAGT-3′, mouse β2 microglobulin; (forward) 5′-CTGACCGGCCTGTATGCTAT-3′ and (reverse) 5′-TCACATGTCTCGATCCCAGT-3′.
3
qRT-PCR Quantification of mRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA extracted from cells was DNase treated (Ambion, Austin, TX) and reverse transcribed using First Strand cDNA Synthesis Kit with oligo(dT)20 (TOYOBO, Osaka, Japan). The resulting cDNA was then used as a template for quantitative RT-PCR using THUNDERBIRD SYBR qPCR Mix (TOYOBO) and a LightCycler 2.0 (Roche). Quantified mRNAs were normalized to the ACTB or GAPDH mRNA level.
4
Total RNA Extraction and Reverse Transcription
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted using Sepasol RNAI Super (Nacalai Tesque, Inc.), as described by the manufacturer's instructions. The extracted total RNA samples were dissolved in TE buffer (10 mM Tris–HCl, pH 8.0, 1 mM EDTA) and stored at −80°C until use. The RNA samples were treated with RQ1 RNase-free DNase mixture (Promega Co., Madison, WI, USA; 1 µg total RNA, 1× DNase buffer, and 1 UDNase in 10 µl) 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 with 1URQ1 DNase Stop Solution (Promega Co.). The concentration of RNA in each sample was measured using Nano Drop Lite (Thermo Fisher Scientific., Waltham, MA, USA). The RNA samples were reverse-transcribed using Rever-Tra Ace (Toyobo Co., Ltd., Osaka, Japan) as per manufacturer's instructions. The reaction mixture (10 µl) comprised 0.5 µg total RNA, 1× reverse transcription buffer (Toyobo Co., Ltd.), 1 µM deoxyribonucleotide triphosphate (dNTP) mixture (Toyobo Co., Ltd.), 5 URNase inhibitor (Toyobo Co. Ltd.), 0.25 µg of oligo (dt) 20 (Toyobo Co., Ltd.), and 50U Rever Tra Ace. The reverse transcription was performed at 42°C for 30 min, followed by heat inactivation at 99°C for 5 min using a programmable thermal controller. Finally, the cDNA samples were stored at −20°C until use.
5
RNA Extraction and Reverse Transcription
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted using Sepasol RNA Super (Nacalai Tesque, Inc., Kyoto, Japan), following the manufacturer's instructions. The extracted total RNA samples were dissolved in TE buffer (10 mM Tris–HCl, pH 8.0, 1 mM EDTA) and stored at −80°C until required. Samples were treated with RQ1 RNase-free DNase mixture (Promega Co., Madison, WI; 1-μg total RNA, 1 × DNase buffer, and 1 unit DNase in 10 μL) on a programmable thermal controller (PTC-100; MJ Research, Waltham, MA), programmed at 37°C for 30 min, followed by incubation at 65°C for 10 min with 1 U RQ1 DNase Stop Solution (Promega Co.). The concentration of RNA in each sample was measured using NanoDrop Lite (Thermo Fisher Scientific., Waltham, MA). The RNA samples were reverse-transcribed using ReverTra Ace (Toyobo Co., Ltd., Osaka, Japan) as per manufacturer's instructions. The reaction mixture (10 μL) comprised 0.5-μg total RNA, 1 × reverse transcription buffer (Toyobo Co., Ltd.), 1-mM deoxyribonucleotide triphosphate (dNTP ) mixture (Toyobo Co., Ltd.), 5 units RNase inhibitor (Toyobo Co. Ltd.), 2.5 pmol oligo (dt) 20 (Toyobo Co., Ltd.), and 50 units ReverTra Ace. Reverse transcription was performed at 42°C for 30 min, followed by heat inactivation at 99°C for 5 min using a programmable thermal controller. Finally, the cDNA samples were stored at −30°C until use.
6
Total RNA Isolation and Reverse Transcription
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from the cells with a RNeasy Mini Kit (Qiagen, Venlo, The Netherlands) and QIAshredder (Qiagen, Venlo, Netherlands), and treated with RQ1 RNase-free DNase (Promega, Madison, WI, USA), according to the manufacturers’ protocols. The resulting sample was subjected to reverse transcription with ReverTra Ace (Toyobo, Osaka, Japan). Briefly, 1 µg of purified total RNA was used in the reaction in the presence of 5 pmol of oligo(dT)20 (Toyobo, Osaka, Japan), and 25 pmol of random primer (Toyobo, Osaka, Japan).
7
Ileum and Cecum RNA Extraction, DNase Treatment, and Reverse Transcription
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
8
Comprehensive RNA Extraction and Reverse Transcription
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted using Sepasol RNA I Super (Nacalai Tesque, Inc. Kyoto, Japan) per the manufacturer's instructions. The extracted total RNA samples were dissolved in TE butter (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and stored at −80°C until further use. The RNA samples were treated with 1URQ1 RNase-free DNase (Promega Co. Madison, WI, USA) in a 10 µl reaction mixture (1 µg total RNA, 1× DNase buffer and 1UDNase) 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 with 1URQ1 DNase Stop Solution (Promega Co.). The concentration of RNA in each sample was measured using a NanoDrop Lite (Thermo Fisher Scientific, Waltham, USA). The RNA samples were 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.), 5U RNase inhibitor (Toyobo Co. Ltd.), 0.25 µg oligo(dT) 20 (Toyobo Co. Ltd.), and 50 UReverTra Ace. Reverse transcription was performed at 42°C for 30 min, followed by heat inactivation at 99°C for 5 min using a programmable thermal controller. The cDNA samples were stored at −20°C until use.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!