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OneScript Plus cDNA Synthesis Kit

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The OneScript Plus cDNA Synthesis Kit is a tool used for the reverse transcription of RNA into complementary DNA (cDNA) for downstream applications. It includes reagents and buffers necessary for the conversion process.

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29 protocols using OneScript Plus cDNA Synthesis Kit

Total RNA was isolated using TRIzol according to the manufacturer's instructions (Fisher Scientific, Pittsburgh, PA). Total RNA was reconstituted in 50 μl of nuclease-free water. Final RNA concentrations were measured by spectroscopy, and cDNA was synthesized using 1 μg of RNA with an OneScript® Plus cDNA Synthesis Kit (abm, Richmond, BC, Canada). Residual genomic DNA was removed by incubation with DNaseI (Thermo Fisher Scientific, Waltham, MA) for 30 min at 37 °C followed by enzyme inactivation with EDTA at 65 °C for 10 min. The resulting cDNA was diluted 1:3 with nuclease-free water to create the template for RT-qPCR.
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Whole larvae from the third instar larval stage or adult thoraxes were briefly washed in PBS and temporarily stored in RNAlater Stabilization solution (Invitrogen AM7020). Total RNA was extracted using RNeasy Mini Kits (Qiagen 74101). The first strand of cDNA was synthesized using the OneScript Plus cDNA Synthesis Kit (Abm G236). qRT-PCR were performed using SensiFast SYBR Lo-ROX Kit (Bioline 94005) on StepOne Real-time PCR System (Applied Biosystems). Primers used are:
cdk8 F CATCCGGGTGTTTCTGTCG
cdk8 R CAGCCCGATGGAACTTAATGAT
cycC F AGTTTCCCTACCGCACCAATC
cycC R ACAATCAAGCAGCAATCCAGG
pink1 FAAGCGAGGCTTTCCCCTAC
pink1 R GCACTACATRGACCACCGATTT
parkin F GAAGCCTCCAAGCCTCTAAATG
parkin R ACGGACTCTTTCTTCATCGGT
opa1 F CAAGCTGCGATACATCGTCC
opa1 R GCAGTCCATCCTTCCATTCC
marf F GAGACGACCACCTTTATCAACG
marf RCCACCTTCATGTGATCCCG
drp1 FACAGCCCACTCGATGATCG
drp1 RAAGCACTTCTTGGTGTGCAG
rp49 FAGCATACAGGCCCAAGATCG
rp49 RTGTTGTCGATACCCTTGGGC
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Imaginal discs from third instar larva were dissected in PBS and temporarily stored in RNAlater Stabilization solution (Invitrogen AM7020). Total RNA was extracted using RNeasy Mini Kits (Qiagen 74101). First strand cDNA was synthesized using OneScript Plus cDNA Synthesis Kit (Abm G236). qRT-PCR were performed using SensiFast SYBR Hi-ROS Kit (Bioline 92005) on StepOne Real-time PCR System (Applied Biosystems).
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Total RNA was extracted from fresh-frozen tissues and cell lines with TRIzol reagent (Invitrogen). cDNAs were synthesized with OneScript Plus Reverse Transcriptase (abm). Quantitative real-time PCR (qPCR) was performed with the OneScript Plus cDNA Synthesis kit (abm). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal normalization reference. The qPCR primers used are listed as follows: TRIM29 forward: AGCATCAGCGACTCTGTGTTG, TRIM29 reverse: GAAGTTGCCTAGTGACTGTCC; PKM1 forward: TGTACCATTGGCCCAGCTTC, PKM reverse: CAGCCACAGGATGTTCTCGT; GATA2 forward: GCCGGGAGTGTGTCAACTG, GATA2 reverse: AGGTGGTGGTTGTCGTCTGA; GAPDH forward: GAGAAGGCTGGGGCTCATTT, GAPDH reverse: AGTGATGGCATGGACTGTGG.
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Pear leaf samples were collected from treated and control plants and immediately frozen at −80 °C. The total RNA was extracted using the Spectrum Plant Total RNA Prep kit (SIGMA Life Science, St. Louis, MO, USA) from 100 mg of tissue. Upon DNAse treatment (ThermoFisher Scientific, Waltham, MA, USA), 1 µg of RNA was retrotranscribed with a OneScript Plus cDNA Synthesis kit (Abm, Vancouver, BC, Canada). Real-time PCR reactions were performed in a CFX Connect Real-Time (Bio-Rad, Hercules, CA, USA) containing 1.6 µL of cDNA, 6 µL of SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA), and 0.4 µL of each primer (10 μM) in a 12 µL final volume. The amplification conditions were 95° for 2 min, 40 cycles at 95 °C/10 s, and 60 °C/30 s. A melting curve analysis from 55 to 95 °C was applied after the amplification protocol. Gene expression was normalized using the EF1α reference gene and analysed by the Livak method [26 (link)] followed by the ANOVA nonparametric Mann-Whitney test. The primers used are shown in Table 1 (PCP sequences were obtained from the Genome Database for Rosaceae, GDR, [27 (link)]).
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To extract total RNA from VSMCs, TRIzol reagent (Tran-szolUp, Transgenbiotech, China) was used following the manufacturer's instructions. Subsequently, the extracted RNA was reverse transcribed using the OneScript Plus cDNA synthesis kit (ABM, Canada) according to the provided instructions.
Expression levels of osteogenic-related (Runt-related transcription factor 2; Runx2 and ALP) and antioxidant system-related genes (Nrf2 and Nqo1) were determined using qRT-PCR. For this analysis, predesigned FAM™labeled TaqMan assays and TaqMan™ fast advanced master mix (Thermo Fisher, 4444557) were employed. The Step One Plus™ real-time PCR system (Thermo Fisher, USA) was used to run the PCR reactions. The mRNA levels of Runx2 (Rn01512298_m1), ALP (Rn01516028_m1), Nrf2 (Rn00582415-m1), and Nqo1 (Rn00566528_m1) were quantified using the 2 -ΔΔCT method, with betaactin (Rn00667869_m1) serving as the reference gene for normalization.
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DNA/RNA were previously extracted from PBMCs using Qiagen’s QIAamp DNA blood mini kit and TRIzol reagent (Gibco, Invitrogen Corporation, Waltham, MA, USA), respectively, as per the manufacturer’s instructions [24 (link)]. The DNA and RNA samples were stored at −20 °C and −80 °C, respectively, until further processing. Approximately 500 ng of RNA was reverse transcribed by using OneScript® Plus cDNA Synthesis Kit, ABM, Canada (Cat#G236), as per the manufacturer’s instructions.
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A549 cells were treated with TD034 at 5 and 10 μM for 24
hr. Western blot was used to check for YAP1 protein level. qRT-PCR
was used to check for YAP1 downstream genes transcription (CTGF and
CYR61). Total RNA was extracted using IBI Isolate Total Extraction
Reagent Kit (IB47602). Two milligrams of RNA was reverse transcribed
using OneScript Plus cDNA Synthesis Kit (ABM G236) according to the
manufacturer’s protocol. Real-time PCR was performed using
BlasTaq 2X qPCR MasterMix (ABM G892) on a QuantStudio 3 Real-Time
PCR System. All qPCR reactions were performed in triplicates. The
list of primers is included in the Supporting Information.
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To remove genomic DNA, samples were DNase-treated according to manufacturer’s instructions (Thermo Scientific, Waltham, MA, USA). Briefly, 1 µg RNA was incubated with 1 U DNase I and reaction buffer (containing MgCl2) at 37°C for 30 min. Subsequently, DNase was inactivated by incubation at 65°C for 10 min in the presence of EDTA, and the DNase-digested RNA was directly used as a template for reverse transcription using the OneScript® Plus cDNA Synthesis Kit (ABM, Richmond, Canada) with oligo(dT) primers.
qPCR was performed on a qTOWER3
G cycler (IST Innuscreen) using the PerfeCTa® Green FastMix® Low ROX (Quantabio). Samples were analyzed in triplicates with 10 ng input cDNA per reaction and 300 nM forward and reverse primers. Data was normalized to the housekeeping gene GAPDH and fold changes calculated using the comparative Ct (ΔΔCt) method. Primer sequences38 (link),39 (link) are listed in Table 1.
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Total RNA was purified using an RNeasy Mini Kit (QIAGEN). RNA concentration was determined using a NanoDrop. 750 ng of RNA was used for reverse transcription reactions using SuperScript III First‐Strand Synthesis SuperMix (Invitrogen). The mRNA levels were then quantified by qPCR using SYBR Premix Ex Taq (TaKaRa) on Roche Light Cycler. Primer sequences: STAT1‐Forward GATGTTTCATTTGCCACCATCCGTTTTC. STAT1‐Reverse GGCGTTTTCCAGAATTTTCCTTTCTTCC. GAPDH‐Forward CCATGTTCGTCATGGGTGTGAACCATG. GAPDH‐Reverse CCACAGCCTTGGCAGCGCCAGTAGAGG. DDX58_Forward CGGCACAGAAGTGTATATTGGATGCATTC. DDX58_Reverse GGAAGCACTTGCTACCTCTTGCTCTTC.
IFIH1_Forward CTGGGACTAACAGCTTCACCTGGTGTTG. IFIH1_Reverse GCATCTGCAATGGCAAACTTCTTGCATG. To measure NF‐κB target expressions, 500 ng of RNA was retro‐transcribed using OneScript Plus cDNA Synthesis Kit (abm, G236) and the following TaqMan assays were used (Thermo Fisher Scientific): IL‐1b, hs00174097_m1; IL‐6, Hs00985639_m1; IL‐8, hs00174103_m1.
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