In brief, 3′-ends of 60 ng of sRNA from each sample were ligated to 5′ pre-adenylated 3′-adaptors (5′- rApp/CTGTAGGCACCATCAAT/3ddC-3′) using truncated T4RNA ligase 2 enzyme (NEB). 3′- adaptor-ligated sRNAs were reverse transcribed with ReverTraAce, reverse transcription polymerase (Toyobo) using an oligonucleotide complementary to the 3′-adaptor (IDT) in a MiniAmp plus Thermal Cycler (Applied Biosystems). RT-qPCR was performed using forward primers for each target piRNA and the oligonucleotide complementary to the 3′-adaptor was used as a reverse primer (Table 5 ). This technique only allows the detection of mature piRNAs. Data were collected in triplicate for each sample on an ABI 7900 Prism qPCR machine and normalized using U6RNA as an internal control. Relative gene-expression levels were calculated using the fold-change method.
Revertra ace reverse
Manufactured by Toyobo
Sourced in Japan
ReverTra Ace reverse is a laboratory equipment product designed for cDNA synthesis. It is a reverse transcriptase enzyme that catalyzes the conversion of RNA into complementary DNA (cDNA). The product's core function is to provide a reliable and efficient tool for researchers to generate cDNA from RNA samples for further molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Miniamp plus thermal cycler, by Thermo Fisher Scientific (1 mentions)
Steponeplus, by Thermo Fisher Scientific (1 mentions)
Oligo dt 15 primer, by Takara Bio (1 mentions)
Isogen 2, by Nippon Gene (1 mentions)
Thunderbird next sybr qpcr mix kit, by Toyobo (1 mentions)
Mastercycler ep realplex2, by Eppendorf (1 mentions)
Rnase free dnase 1, by Takara Bio (1 mentions)
Revertra ace qpcr rt master mix, by Toyobo (1 mentions)
Trizol reagent, by Tiangen Biotech (1 mentions)
Sybr green realtime pcr master mix, by Toyobo (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Purelink ffpe total rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Stepone software, by Thermo Fisher Scientific (1 mentions)
Thunderbird probe qpcr mix, by Toyobo (1 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (1 mentions)
4 protocols using revertra ace reverse
1
Detecting Mature piRNAs via RT-qPCR
2
RNA Extraction and qPCR Analysis Protocol
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An RNA extraction solution, Isogen II (Nippon Gene Co., Tokyo, Japan), was used to isolate total RNA from tissue pieces of the lung stored in RNA Save solution. The cDNA was synthesized by incubating 2–4 µg of total RNA with 100 U of ReverTra Ace reverse transcriptase (Toyobo Co., Osaka, Japan) with a mixture of 20 pmol random hexamers pdN6 and 5 pmol oligo-dT(15) primers (Takara Bio Inc., Kusatsu, Japan). A qPCR instrument, StepOnePlus (Applied Biosystems/Life Technologies Co., Carlsbad, CA, USA), was employed for cDNA measurement with Thunderbird Next Sybr qPCR Mix Kit (Toyobo Co.). PCR fragments for each cDNA were prepared separately and purified by gel electrophoresis before quantitative analysis. The DNA sequences were confirmed by Fasmac Co., Ltd. (Atsugi, Japan). The extracted DNA fragments were used as standards for quantification. PCR conditions were 2 min of initial denaturation followed by 40 cycles of 5 s at 95 °C and 35 s at 60 °C. The measured mRNA levels were normalized with reference to the β-actin mRNA levels. Specific primer sets for genes are listed in Table 1 .
3
RNA Extraction and RT-qPCR Analysis of Mitochondrial Transcripts
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Total RNA was extracted from the samples using TRIzol reagent (Tiangen) according to previous Feng et al. (2009) (link), and DNA was removed by treatment with RNase-Free DNase I (Takara). Using ReverTra Ace reverse transcriptase (Toyobo), RNA was reverse-transcribed to complementary DNA using the random primers provided in the kit (ReverTra Ace qPCR RT Master Mix, Toyobo). Amplification of mitochondrial transcripts was performed using primers as described previously (Chen et al., 2017 (link)). Quantitative RT-PCR was performed using three independent sets of RNA with Ubiquitin as the reference gene. Primers for the mitochondrial introns were designed as described previously with some modifications (Qi et al., 2017b (link)) (Supplementary Table S2 at JXB online). Quantitative RT-PCR was performed with SYBR Green Real-Time PCR Master Mix (Toyobo) using a Mastercycler ep realplex 2 (Eppendorf) according to the manufacturer’s protocol.
4
FFPE RNA Extraction and qPCR Analysis
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Five to eight sections of 10–15 μm thickness were sliced from the FFPE blocks. The first slice was discarded to avoid contamination. Total RNAs were extracted from the FFPE tissue sections using PureLink FFPE Total RNA isolation Kit (Invitrogen Life Technologies, Carlsbad, CA). Total RNA (1 μg) was reverse-transcribed using the ReverTra Ace reverse transcription-quantitative polymerase chain reaction (RT qPCR) Master Mix (TOYOBO, Osaka, Japan). Quantitative real-time PCR was performed using the Thunderbird Probe qPCR Mix (TOYOBO) and the StepOne Real-Time PCR system (Applied Biosystems, Foster City, CA). The conditions for PCR were 95°C for 1 min, and then 40 cycles of 95°C for 15 sec (denaturation) and 60°C for 1 min (annealing extension). Primers and fluorogenic probes for CCL21 (Hs00171076_m1), CCL19 (Hs00355524_g1), CCR7 (Hs01013469_m1), and β2-microglobulin (Hs00984230_m1) were obtained from TaqMan Gene Expression Assays (Applied Biosystems). Gene expression was quantified using StepOne software (Applied Biosystems). To avoid sampling bias, we first confirmed that there were no significant differences in the expression levels of β2-microglobulin gene in the samples, and then we examined the expression of CCL21, CCL19, and CCR7.
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