Total RNA, inclusive of the small RNA fraction, was prepared from homogenized tissues and cultured cells with Trizol reagent as recommended by the manufacturers (Invitrogen). For circ_0018289 and mRNA quantification, cDNA was randomly primed with ReverTra Ace RT Kit (Toyobo, Tokyo, Japan) in a 25-μL reaction containing 500 ng of extracted RNA. qRT-PCR with iQ SYBR Green (Bio-Rad, Munich, Germany) and amplification primers (shown in Supplementary Table 1 ) was done on a PCR machine (Rotorgene 6000, Qiagen) with the following conditions: after a denaturation time of 10 min at 95 °C, 40 cycles at 95 °C for 20 s and at 60 °C for 1 min. For miR-183-5p quantification, cDNA preparation and qRT-PCR were carried out as above, using miScript RT Kit and SYBR Green Kit (all from Qiagen), respectively. Fold changes in gene and miRNA expression were determined by the 2−ΔΔCt method, normalizing the results to normal controls and expression of β-actin or U6. ΔCt was calculated by subtracting the Ct values of β-actin or U6 from the Ct values of the gene of interest. ΔΔCt was then calculated by subtracting ΔCt value of the control from ΔCt of the sample.
Revertra ace rt kit
Manufactured by Toyobo
Sourced in Japan
The ReverTra Ace RT Kit is a laboratory product designed for reverse transcription. It provides the necessary components for converting RNA into complementary DNA (cDNA) in a single-tube reaction. The kit includes reverse transcriptase enzyme, reaction buffer, and other essential reagents required for the reverse transcription process. The product's core function is to facilitate the conversion of RNA to cDNA, which is a crucial step in various molecular biology and gene expression analyses.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Steponeplus system, by Thermo Fisher Scientific (2 mentions)
Mir x mirna first strand cdna synthesis kit, by Takara Bio (2 mentions)
Blend taq, by Toyobo (2 mentions)
Rotor gene 6000, by Qiagen (1 mentions)
Sybr green kit, by Qiagen (1 mentions)
Miscript rt kit, by Qiagen (1 mentions)
Iq sybr green, by Bio-Rad (1 mentions)
Ex taq, by Takara Bio (1 mentions)
Redsafe, by iNtRON Biotechnology (1 mentions)
Sybr green pcr master mix, by Toyobo (1 mentions)
Quantstudio 6 flex pcr system, by Thermo Fisher Scientific (1 mentions)
Kapa sybr fast qpcr kit, by Roche (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Taqman mirna assay kit, by Thermo Fisher Scientific (1 mentions)
Taqman mirna rt kit, by Thermo Fisher Scientific (1 mentions)
Qrt pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr qpcr mix, by Toyobo (1 mentions)
Sybr green 1 reaction mix, by Toyobo (1 mentions)
Applied biosystems quantstudio 5 real time detection system, by Thermo Fisher Scientific (1 mentions)
12 protocols using revertra ace rt kit
1
Quantification of circular RNA and mRNA
2
Profiling Metallopeptidase Expression in T. vaginalis
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Total RNA was extracted from T. vaginalis T016 isolate using Trizol Reagent (Invitrogen, Carlsbad, California, USA), and RNA concentrations were determined spectrophotometrically. RNA (1 µg) was reverse transcribed using the ReverTra Ace RT kit (Toyobo, Osaka, Japan). Following reverse transcription, cDNA was amplified by PCR using Ex Taq (Takara, Tokyo, Japan) under the following conditions: 5 min at 94℃, followed by 30 cycles of 30 sec at 98℃, 30 sec at 57℃, and 1 min at 72℃. The final elongation step was continued for 10 min at 72℃. The primer sequences were used as follows: GP63, MG aminopeptidase P-like metallopeptidase (MP50), M41 FtsH endopeptidase-like metallopeptidase (TVAG_243780.2), oligopeptidase A-like metallopeptidase (TVAG_477180), zinc carboxypeptidase-like metallopeptidase (TVAG_075740), and aspartyl aminopeptidase-like metallopeptidase (TVAG_392410) (Table 1 ). PCR products were electrophoresed through a 2% agarose gel and visualized with RedSafe (iNtRON Biotechnology, Seoul, Korea).
3
Quantifying ZIKV RNA Levels by qRT-PCR
4
Quantification of mRNA and miRNA Transcripts
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Total RNAs were isolated from cell lines using Trizol reagent (Invitrogen, CA). For the analysis of mRNA, complementary DNA (cDNA) was synthesized by reverse transcription using a ReverTra Ace® RT Kit (Toyobo, Japan). For miRNA analysis, cDNA was prepared using the MiR-X™ miRNA First-Strand cDNA synthesis kit (Clonetech, CA) according to the manufacturer’s instructions. The relative abundance of each transcript was assessed by real-time quantitative polymerase chain reaction (RT-qPCR) using the Kapa SYBR Fast qPCR kit (Kapa Biosystems, MA) and specific primer sets on the StepOne Plus™ system (Applied Biosystems, CA). Primer sequences are listed in Table 1 .
5
Gene Expression Analysis of TCTN2, DUSP1, and miR-125a-5p
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Total RNA was prepared using the RNeasy Mini Kit as recommended by the manufacturers (Qiagen, Crawley, UK). To measure TCTN2 and DUSP1 expression, single-stranded cDNA was made in a 25 μL reaction using the ReverTra Ace RT Kit (Toyobo, Tokyo, Japan) from 2 μg of RNA; qRT-PCR was conducted using SYBR® qPCR Mix (Toyobo) and designed primers for mouse (TCTN2-forward: 5′-GCCCCAACTTCTGTACCCTC-3′, TCTN2-reverse: 5′-GTAGATGGCAGCTGAGTCCC-3′, DUSP1-forward: 5′-AGTGCCTATCACGCTTCTCG-3′, DUSP1-reverse: 5′-CCTCCACAGGGATGCTCTTG -3′); and mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (forward: 5′-CCCTTAAGAGGGATGCTGCC-3′, reverse: 5′-ACTGTGCCGTTGAATTTGCC-3′) was used as a reference gene. To quantify miR-125a-5p, 1 μg of RNA was converted to cDNA by the TaqMan miRNA RT Kit (Applied Biosystems, Toronto, Canada); qRT-PCR was done using the TaqMan miRNA Assay Kit (Applied Biosystems) with primers for mouse miR-125a-5p (forward: 5′-GCCGAGTTCCCAGAGTCCCT-3′, reverse: 5′-CTCAACTGGTGTCGTGGA-3′); and the mouse U6 severed as a housekeeping gene for normalization with specific primers (forward: 5′-CGAATCCGAACCTTTCCCCA-3′, reverse: 5′-TTTGGAAAGAGGCCATGCGG -3′). All reactions were run on a qRT-PCR System (Applied Biosystems) in triplicate and analyzed using the comparative Ct method (2−ΔΔCt).
6
Transcriptomic Analysis of Striatal Neurons
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After the whole-cell recording, the contents of the cell were aspirated into the recording pipette and harvested in a sampling tube. The collected samples were reverse-transcribed using a ReverTra Ace RT kit (TOYOBO) and amplified with Blend Taq (TOYOBO). The oligonucleotide primers used were 5'- CCCAGGCGACATCAATTT -3' and 5'-TCTCCCAGATTTTGAAAGAAGG -3' for proenkephalin (Penk); 5'-CCAGGGACAAAGCAGTAAGC -3' and 5'-CGCCATTCTGACTCACTTGTT -3' for prodynorphin (Pdyn); and 5'-CCGCTGATCCTTCCCGATAC -3' and 5'-CGACGTTGGCTGTGAACTTG -3' for enolase 2 (Eno2) as a neuronal marker. The PCR products were analyzed using agarose gel electrophoresis. Pdyn-positive neurons were considered to be direct pathway MSNs (dMSNs), and Pdyn-negative and Penk-positive neurons were considered to be indirect pathway MSNs (iMSNs; Fig. 2A,B ).
7
Single-cell RT-PCR Analysis of Neuronal Markers
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Single-cell RT-PCR was performed as previously described10 (link). After the whole-cell recording, the contents of the cell were aspirated into the recording pipette and harvested in a sampling tube. The collected samples were reverse-transcribed using a ReverTra Ace RT kit (TOYOBO, Tokyo, Japan) and amplified with Blend Taq (TOYOBO, Tokyo, Japan). The oligonucleotide primers used were 5′-TAGGCTTAGCGTCTCTGGGA-3′ and 5′-AAGGCCGAACTCGATTGTGA-3′ for Tph2; 5′-GGCCTGAAGATCTGTGGCTT-3′ and 5′-CAGAACCTTGGTGGAGCGAT-3′) for Gad1; 5′-ATGCAGAGCTGCAACCAGAT-3′ and 5′-GCCTCAAACCCAGTAGTCCC-3′ for Gad2; 5′-CCGCTGATCCTTCCCGATAC-3′ and 5′-CGACGTTGGCTGTGAACTTG-3′ for Eno2 as a neuronal marker. PCR products were analyzed using agarose gel electrophoresis. Only when Tph2 mRNA expression was detected, the data was used for analysis (Fig. 1a ).
8
Quantifying Viral Gene Expression
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To determine the effects on different inhibitors on virus replication, viral gene transcriptions were detected by qRT-PCR. Total RNA from SGIV-infected cells were isolated using the cell total RNA isolation kit (Foregene) according to manufacturer’s instructions. The RNA was reverse transcribed using ReverTra Ace RT Kit (Toyobo). Amplification was examined using SYBR Green I Reaction Mix (Toyobo) in an Applied Biosystems QuantStudio 5 Real Time Detection System (Thermo Fisher). Each assay was carried out under the following cycling conditions: 95 °C for 5 min for activation, followed by 40 cycles at 95 °C for 5 s, 60 °C for 10 s, and 72 °C for 15 s. The primers used were listed in Table 1 . The expression levels of target genes were standardized to β-actin and calculated with the 2−△△CT method. The data were indicated as mean ± SD and shown from one representative experiment carried out in triplicate. Statistical significance was determined with Student’s t-test and the statistical significance was set at p < 0.05.
9
Quantifying Gene Expression in Lung Tissue
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Total RNA was extracted from lung tissue samples and reverse transcribed into cDNA using the ReverTra Ace™ RT kit (TOYOBO). Gene expression was quantified using real-time quantitative PCR (RT-qPCR), using the 2-ΔΔCt method to determine the relative expression levels. Primer sequences can be found in the supplementary materials (Additional file 2 : Table S2 ).
10
Quantitative Analysis of RNA Expression
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We prepared total RNA from cultured cells and tissue samples with peqGOLD total RNA Kit (PeqLab, Erlangen, Germany) and quantified it using Agilent 2100 Bioanalyzer (Agilent Technologies, Stockport, UK). For analysis of circ_MACF1 and mRNA, cDNA was randomly or oligo(dT) primed from 1–2 µg of total RNA using ReverTra Ace RT Kit as per the manufacturing instructions (Toyobo, Tokyo, Japan). For analysis of miR-942-5p, 200 ng of RNA was conversed to cDNA using BON-miR miRNA 1st-strand cDNA Synthesis Kit (Bonyakhteh, Tehran, Iran) based on the accompanying recommendations. Synthesized cDNA was amplified by qRT-PCR using Thunderbird SYBR® qPCR Mix (Toyobo) with specific primers (Additional file 3 : Table S1) on the MyiQ Detection System (Bio-Rad, Gladesville, NSW, Australia). Fold changes were expressed as a corrected value obtained by dividing the expression level by that for β-actin or U6 and calculated under usage of the 2−ΔΔCt method [17 (link)].
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