Chicken sperm total RNA was isolated and purified using the RNAzol method described by Shafeeque et al.53 (link). Sperm RNA quality and concentration were assessed using a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). The absorbance ratios at 260/280 and concentrations of chicken total sperm RNA in the control and plasma treatment group were shown in Supplementary Table S4 . cDNA synthesis was performed using TOPscriptTM RT DryMIX (dT18) (Enzynomics, Daejeon, Republic of Korea). RT-PCR analysis was performed using Prime Taq Premix (2×) (GENETBIO, Yuseong-gu, Daejeon, Republic of Korea), and EvaGreen Dye (Biotium, Hayward, CA, USA) according to the manufacturer’s instructions. Primer sequences for RT-PCR are shown in Supplementary Table S5 . mRNA relative expression levels were normalized to the housekeeping gene (β-actin) and calculated using the 2−ΔΔCT method.
Topscript rt drymix dt18
Manufactured by Enzynomics
TOPscriptTM RT DryMIX (dT18) is a ready-to-use reverse transcription reaction mixture for cDNA synthesis from RNA templates. It contains all the necessary components, including reverse transcriptase and oligo(dT)18 primer, in a convenient dried format.
Automatically generated - may contain errors
Lab products found in correlation
Evagreen dye, by Biotium (2 mentions)
Prime taq premix 2, by GeNet Bio (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Cfx96 qpcr system, by Bio-Rad (1 mentions)
Cfx manager 3, by Bio-Rad (1 mentions)
Topreal qpcr 2x premix sybr green with low rox, by Enzynomics (1 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (1 mentions)
Real time pcr master mix, by Biofact (1 mentions)
4 protocols using topscript rt drymix dt18
1
Chicken Sperm RNA Isolation and Analysis
2
Quantitative RT-PCR Gene Expression Analysis
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For qRT-PCR, cDNAs were synthesized using total RNA with TOPscriptTM RT DryMIX (dT18) (Enzynomics, Daejeon, Korea), according to the manufacturer’s instructions. The qRT-PCR was performed using the CFX96 qPCR system (Bio-Rad) and TOPreal™ qPCR 2X PreMIX (SYBR Green with low ROX, Enzynomics, Daejeon, Korea). The N. salina gene encoding actin served as an internal control. The primers used for qRT-PCR are listed in the Supplementary Table S1
ΔCt = Target Ct mean – actin gene Ct,
ΔΔCt = Sample ΔCt mean - Reference ΔCt mean,
2^(-ΔΔCt) = fold difference.
ΔCt = Target Ct mean – actin gene Ct,
ΔΔCt = Sample ΔCt mean - Reference ΔCt mean,
2^(-ΔΔCt) = fold difference.
3
Investigating mRNA Expression in Chickens
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Total RNA was isolated and purified from skeletal muscles and thyroid glands of 90-day-old chickens using TRIzol Reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). cDNA synthesis was performed using TOPscriptTM RT DryMIX (dT18) (Enzynomics, Daejeon, Korea). RT-PCR analysis was performed using Prime Taq Premix (2×) (GENETBIO, Yuseong-gu, Daejeon, Korea), and EvaGreen Dye (Biotium, Hayward, CA, USA) according to the manufacturer’s instructions. Primer sequences for RT-PCR are shown in Table 3 . mRNA relative expression levels were normalized to the housekeeping gene (β-actin) and calculated using the 2−ΔΔCt method.
4
Quantification of IbFAD8 Gene Expression
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Total RNA was extracted from frozen ground tissues of sweetpotato plants using the TRIzol Reagent (Invitrogen, MA, United States), according to the manufacturer’s instructions. Then, cDNA was synthesized from the isolated RNA using TOPscriptTM RT DryMIX (dT18) (Enzynomics, Daejeon, South Korea). To quantify gene expression levels, qRT-PCR was conducted on a Bio-Rad CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, United States) using 2X Real-Time PCR Master Mix and EvaGreenTM dye (BioFACT, Daejeon, South Korea). The conditions for the qRT-PCR was as follows: initial denaturation for 15 min at 95°C, followed by 45 cycles of 95°C for 20 s, 58°C for 40 s, and 72°C for 20 s. Relative gene expression were calculated using the comparative delta-delta Ct method, and the Ubiquitin gene was used as an internal control for the normalization of IbFAD8 expression levels. The transcript levels were expressed as relative values, which the lowest expression was valued as 1. The sequences of primers used for qRT-PCR are listed in Supplementary Table 1 .
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