For quantitative detection of pre-miR-128, qRT-PCR was designed according to the previously described method47 (link)48 (link). Briefly, total RNA was prepared by TRIzol reagent (Invitrogen). RNA was processed to reverse transcriptase reactions using reverse transcriptase kit (ReverTra Ace, Toyobo). Real time PCR using SYBR Green Realtime PCR Master Mix (ReverTra Ace, Toyobo) was carried out in triplicate in an in a 7500 Fast Real-Time PCR System (Applied Biosystems) according to the manufacturer’s instructions. The sequences of rat pre-miR-128 primers were Forward-1: 5′-CTTTCATTCTTGGGCTCTTTG-3′; Forward-2: 5′-TGAGCTGTTGGATTCGGGGCC-3′; Reverse: 5′-GAAGCAGCTGAAAAAGAGACC-3′.
Revertra ace
Manufactured by Toyobo
Sourced in Japan, United States, China
ReverTra Ace is a reverse transcription kit designed for high-quality cDNA synthesis. It facilitates the conversion of RNA to cDNA, which is a crucial step in various molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (211 mentions)
Thunderbird sybr qpcr mix, by Toyobo (106 mentions)
Rneasy mini kit, by Qiagen (99 mentions)
Trizol, by Thermo Fisher Scientific (90 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (83 mentions)
Isogen, by Nippon Gene (39 mentions)
Sybr premix ex taq 2, by Takara Bio (36 mentions)
Isogen 2, by Nippon Gene (31 mentions)
Sepasol rna 1 super g, by Nacalai Tesque (28 mentions)
Oligo dt, by Takara Bio (27 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (26 mentions)
Sybr premix ex taq, by Takara Bio (24 mentions)
Steponeplus, by Thermo Fisher Scientific (23 mentions)
Rneasy plant mini kit, by Qiagen (22 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (22 mentions)
Lightcycler 480, by Roche (22 mentions)
Dnase 1, by Takara Bio (21 mentions)
Rneasy plus mini kit, by Qiagen (21 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (21 mentions)
Kapa sybr fast qpcr kit, by Roche (18 mentions)
1 031 protocols using revertra ace
1
Quantitative Detection of Pre-miR-128 by qRT-PCR
2
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.
3
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.
4
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.
5
Quantification and Qualitative Analysis of DINE mRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from embryonic spinal cords using the RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. For quantification of DINE mRNA expression, total RNA (1 μg aliquots) was converted to cDNA by ReverTra Ace (Toyobo, Osaka, Japan) and an oligo (dT) primer. The resultant cDNA was diluted 1:50 with distilled water. The quantitative PCR (qPCR) procedures were performed as previously described [24 (link)]. TaqMan Gene Expression Assays (Applied Biosystems, Waltham, MA, USA) for mouse actin beta (ACTB) (Mm00607939_s1) and ECEL1 (Mm00469610_m1) were used for specific target amplification. Relative mRNA expression was calculated by using the comparative cycle threshold (CT) method, and then normalized to endogenous ACTB mRNA expression for each sample. The CT value was obtained from the amplification plot with the aid of SDS software (Applied Biosystems).
For qualitative analysis of the mutant DINE transcript, total RNA (1 μg aliquots) was converted to cDNA using ReverTra Ace (Toyobo, Osaka, Japan) with random primers. The cDNA was amplified using the following specific primers: forward 5′-CCACCCTGTATGACCCAGAC-3′, reverse 5′-ATAGAGGCGAACGATGCACT-3′.
For qualitative analysis of the mutant DINE transcript, total RNA (1 μg aliquots) was converted to cDNA using ReverTra Ace (Toyobo, Osaka, Japan) with random primers. The cDNA was amplified using the following specific primers: forward 5′-CCACCCTGTATGACCCAGAC-3′, reverse 5′-ATAGAGGCGAACGATGCACT-3′.
6
Quantifying Transcripts in Transgenic Plants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA isolated from leaf and floral tissues of T2 plants were analyzed. Following manufacturer protocol, 1 μg of total RNA and an oligo dT20 primer were used for reverse transcription (ReverTra Ace-á, Toyobo, Japan). Transcript levels of NtCHI, NtCHS, NtF3H, NtDFR, NtANS, and ACTIN were measured using a StepOnePlus Real-Time PCR system (Thermo Fisher Scientific, Waltham, USA) (Ai et al. 2016). The primers and PCR conditions for the detected genes are listed in Table 1 . Five samples per line were used, and the analysis was repeated three times.
7
Quantifying Lignin and Ethylene Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from 100 mg stem segments from the bending zones, which were sampled at 3, 6, and 9 DAT using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The cDNA was synthesized from 1 µg of total RNA using an oligo dT20 primer and a reverse transcription kit (ReverTra Ace-á, Toyobo, Japan). The mRNA levels of the lignin biosynthetic genes (PAL (DQ866660.1) and 4CL (Y15607.1)) were analyzed at 3, 6, and 9 DAT, but those of ethylene biosynthetic genes (ACS1 (AF083814.2), ACO1 (AY333925.1), and ACO2 (AY333926.1)) were analyzed only at 6 DAT, using the StepOnePlus Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, United States). Relative gene expression was calculated using the quantitative-comparative method CT (ΔΔCT). The primers and PCR conditions used for this analysis were listed in Additional file 1 : Table S1. Three independent bending zones were used for each analysis and analysis was performed three times.
8
Differential Expression of Carnation Ethylene and Senescence Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from 100 mg of petals using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The cDNA was synthesized from 1 μg of the total RNA with an oligo dT20 primer using a reverse transcription kit (ReverTra Ace-á, Toyobo, Japan). Transcript levels of ethylene production-related genes (DcACO1 and DcACS1), and petal senescence-related gene (cysteine proteinase inhibitor gene; DcCPI) were measured using a StepOnePlus Real-Time PCR system (Thermo Fisher Scientific, Waltham, USA) [18 (link)]. To confirm the amount of template RNA, a fragment of carnation actin (DcACT) was used as the internal control. The primers and PCR conditions for the detected genes are listed in (Additional file 1 : Table S1). Three samples per treatment were used, and the analysis was repeated three times.
9
Carnation Petal Senescence Transcripts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from 100 mg of petals collected on days 3, 6, 10, and 14 using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The cDNA was synthesized from 1 μg total RNA using an oligo dT20 primer and a reverse transcription kit (ReverTra Ace-á, Toyobo, Japan). The transcript levels of 1-aminocyclopropane-1-carboxylate oxidase (DcACO1), 1-aminocyclopropane-1-carboxylate synthase (DcACS1), and the petal senescence-resistance genes (cysteine proteinase inhibitor gene DcCPi) were measured using a StepOnePlus Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, United States). To confirm the amount of template RNA, fragments of the carnation reference genes (DcUbq4-5 and DcACT) were used as internal controls. The primers and PCR conditions used for detecting the expression levels of these genes are listed in Table 1 . The method used to calculate relative gene expression was quantitation–comparative method CT (ΔΔCT). Five samples per treatment were used, and each analysis was repeated three times.
10
Analyzing Lignin and Ethylene Genes in Bending Stems
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from 100mg of stem segments from the bending zone, which were sampled at 3, 6, and 9 DAT, using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). cDNA was synthesized from 1μg of total RNA using an oligo dT20 primer and a reverse transcription kit (ReverTra Ace-á, Toyobo, Japan). The mRNA levels of lignin biosynthetic genes [PAL (DQ866660.1) and 4CL (Y15607.1)] were analyzed using the StepOnePlus Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, United States). Similarly, the transcript levels of ethylene biosynthetic genes [ACS1 (AF083814.2), ACO1 (AY333925.1), and ACO2 (AY333926.1)] were detected from the bending zones of stems sampled at 6 DAT. The primers and PCR conditions used for this analysis are listed in Supplementary Table 1 . Three independent bending zones were used for each analysis, with three replicates.
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!