Chickens at 20 days of age were allotted randomly to three groups of ten each. Groups 1 and 2 were inoculated intranasally with 100 μl of the GD strain at 106.2 50% egg infective doses (EID50), or the same dose of F48E9 respectively. Group 3 was inoculated with 100 μl of phosphate‐buffered saline served as a control. Five birds from each group were slaughtered at 24 and 48 hr postinfection (hpi) respectively. Tissue samples of lung, trachea, proventriculus, kidney, cecal tonsil, liver, spleen, Harderian gland, bone marrow, and brain were collected and used for quantitative RT‐PCR analyses of host genes and NDV. The mRNA expression levels of the target genes were measured using One‐step Real‐time PrimeScript RT‐PCR (Takara Biotechnology, Dalian, China) with specific primers (Supporting Information Table S1 ), as described previously (Li et al., 2015 ; Xu et al., 2016 ). All amplifications were performed in triplicate. The mRNA levels of each target gene were normalized to the levels of 18S rRNA in the same samples.
One step real time primescript rt pcr
Manufactured by Takara Bio
Sourced in China
The One‐step Real‐time PrimeScript RT‐PCR is a reagent kit designed for reverse transcription and real‐time PCR in a single reaction. It enables the detection and quantification of RNA targets.
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Lab products found in correlation
2 protocols using one step real time primescript rt pcr
1
Quantitative Analysis of Host Response to Avian Influenza
2
Quantifying FAdV Viral Loads and Gene Expression
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All tissue samples were tested for FAdV viral load using RT-qPCR as described previously (Li et al., 2015 (link); Zhao et al., 2020 ). The primer used in this study has been described previously (Zhao et al., 2020 ). All samples were analyzed in triplicate.
At each time point, total RNA was extracted from tissue samples using TRIzol reagent (Invitrogen, Beijing, PR China) according to the manufacturer's protocol. The mRNA expression levels of the target genes were measured using One-step Real-time PrimeScript RT-PCR (Takara Biotechnology, Dalian, China) with specific primers, as described previously (Li et al., 2015 (link); Xu et al., 2016 (link); Zhang et al., 2019 (link)). The mRNA levels of the selected genes were quantified by using standard curves generated by a serial dilution of plasmids as described previously (Zhang et al., 2019 (link)). 18S rRNA was used as the reference gene. The data for each target gene are expressed as the copy number of each target cDNA normalized to that of the reference gene in the same samples, as described previously (Li et al., 2015 (link)).
At each time point, total RNA was extracted from tissue samples using TRIzol reagent (Invitrogen, Beijing, PR China) according to the manufacturer's protocol. The mRNA expression levels of the target genes were measured using One-step Real-time PrimeScript RT-PCR (Takara Biotechnology, Dalian, China) with specific primers, as described previously (Li et al., 2015 (link); Xu et al., 2016 (link); Zhang et al., 2019 (link)). The mRNA levels of the selected genes were quantified by using standard curves generated by a serial dilution of plasmids as described previously (Zhang et al., 2019 (link)). 18S rRNA was used as the reference gene. The data for each target gene are expressed as the copy number of each target cDNA normalized to that of the reference gene in the same samples, as described previously (Li et al., 2015 (link)).
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