Total RNAs from mouse tissues were isolated using Trizol reagent (Life Technologies Inc., Gaithersburg, USA) according to the manufacturer’s instructions. The first-strand cDNAs were synthesized from 2-μg of total RNAs with oligo (dT) primer and random six mer primer using PrimerScript RTase (TaKaRa, Dalian, China) at 37°C for 15 min. cDNAs were amplified using specific sets of primers listed in Supplementary Table S1
Primerscript rtase
Manufactured by Takara Bio
Sourced in China
PrimerScript RTase is a reverse transcriptase enzyme used for the synthesis of first-strand cDNA from RNA templates. It exhibits high thermal stability and is capable of synthesizing cDNA from a wide range of RNA templates, including mRNA, total RNA, and viral RNA.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler 480, by Roche (2 mentions)
Sybr premix ex taq kit, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Mastercycler ep realplex, by Eppendorf (1 mentions)
Sybr premix ex taqtm 2, by Takara Bio (1 mentions)
Rnaiso plus reagent, by Takara Bio (1 mentions)
Trnzol a reagent, by Tiangen Biotech (1 mentions)
4 protocols using primerscript rtase
1
Quantitative Real-Time PCR Analysis
2
Quantitative Gene Expression Analysis in Termites
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Total RNA was extracted using RNAiso Plus reagent (TaKaRa Bio. Inc., Japan) from the head-thoraxes of the adultoid reproductives, brachypterous neotenics and alate reproductives that had just moulted from the last instar nymphs (n = 5). cDNA was synthesised from 50 ng of mRNA. cDNA for qPCR was synthesized by PrimerScript RTase (Takara Bio. Inc., Japan). The quantitative reaction was performed on a LightCycler 480 software release 1.2.0.0625 (Roche Diagnostics, Switzerland) using SYBR Premix Ex TaqTM II (TaKaRa Bio. Inc., Japan). All reactions in the q-PCR system were normalized using the Ct values corresponding to the actin levels according to a study of reliable reference genes for expression studies using q-PCR in Odontotermes formosanus6 (link). The relative gene expressions were calculated using the 2−∆∆Ct method26 (link). All qRT-PCR experiments were repeated in three biological and three technical replications. The non-parametric Kruskal-Wallis test combined with post hoc Dunn’s multiple comparisons test was used to compare statistical differences among ARs, ANs and BNs.
3
Transcriptome Analysis of Termite Females
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The total RNA of ten eggs in R. aculabialis and R. flaviceps female–female colony types was extracted using TRNzol-A+ Reagent (Tiangen, Beijing, China), according to the manufacturer’s protocol. RNA quality was verified using a spectrophotometer (Eppendorf BioPhotometer, Shanghai, China). cDNA for qPCR was synthesized using Primer Script RTase (TaKara Bio. Inc., Hong Kong, China). The quantitative reaction was performed with LightCycler 480 software release 1.2.0.0625 (Roche Diagnostics, Indianapolis, IN, USA) using SYBR Premix Ex TaqTMⅡ (TaKara Bio. Inc., Baori Medical Biotechnology Beijing, China). All reactions in the qPCR system were normalized using the Ct values corresponding to the actin levels according to a study of reliable reference genes for expression studies using qPCR in Odontotermes formosanus [35 (link)]. The relative gene expressions were calculated using the 2−∆∆Ct method [36 (link)] and the data were analyzed with SPSS 18.0. All qPCR experiments were repeated for three technical replications. The fragment size of the primer was between 100 and 250 bp, with details presented in Table 1 .
4
Profiling Vitis Jasmonate Signaling
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Total RNA was extracted from V. quinquangularis cv. “Shang-24” according to the protocol of Huang et al., 2016 [54 (link)]. First strand cDNA was synthesized from two μg of total RNA with the Primer Script™ RTase according to the supplied protocol (TaKaRa Bio Inc., Dalian, China). cDNA was amplified using 2× Taq PCR Master Mix (Bio Sci Biotech, Hangzhou, China) and oligonucleotide primers designed to specifically amplify the VqJAZ or VvJAZ transcripts (Table S1 ). Grapevine ACTIN1 was used as a reference gene. Reactions were conducted with the following parameters: 94 °C for eight minutes, followed by 30–40 cycles of 92 °C for 30 s, 58–64 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for eight minutes. PCR products were separated on a 1% (w/v) agarose gel and imaged under UV light to analyze gene expression. All of the reactions were repeated three times.
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