Quantitative RT-PCR was performed to assess viral mRNA transcripts from infected ferret tracheal and lung samples used for sequencing. Total RNAs were treated with DNase using DNA-free DNase Treatment and Removal Reagents (Ambion, Inc, Austin, TX). cDNAs from total RNAs were generated using the QuantiTect reverse transcription kit (Qiagen Inc.). A custom-designed TaqMan gene expression assay for influenza Matrix (M) sequence (MPCONS2010), with primers that have complete homology with both 1918 and CA04 M sequences, was ordered from Applied Biosystems, Inc. Taqman experiments were performed on the ABI 7500 Real-Time PCR System platform and each sample was run in quadruplicate. Ribosomal RNA (18S) was used as endogenous control to normalize quantification of each target within tissues using Applied Biosystems Sequence Detections Software version 1.3. The relative amount of viral mRNA (log 10) is presented in the final results.
Abi 7500 real time pcr system platform
Manufactured by Thermo Fisher Scientific
The ABI 7500 Real-Time PCR System is a platform designed for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. It is capable of performing real-time detection and quantification of nucleic acid sequences.
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Lab products found in correlation
Dnase treatment and removal reagent, by Thermo Fisher Scientific (2 mentions)
Taqman gene expression assay for influenza matrix, by Thermo Fisher Scientific (2 mentions)
Sequence detections software version 1, by Thermo Fisher Scientific (2 mentions)
Quantitect reverse transcription kit, by Qiagen (2 mentions)
2 protocols using abi 7500 real time pcr system platform
1
Quantitative RT-PCR for Influenza Virus
2
Quantitative RT-PCR for Influenza Virus
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Quantitative RT-PCR was performed to assess viral mRNA transcripts from infected ferret tracheal and lung samples used for sequencing. Total RNAs were treated with DNase using DNA-free DNase Treatment and Removal Reagents (Ambion, Inc, Austin, TX). cDNAs from total RNAs were generated using the QuantiTect reverse transcription kit (Qiagen Inc.). A custom-designed TaqMan gene expression assay for influenza Matrix (M) sequence (MPCONS2010), with primers that have complete homology with both 1918 and CA04 M sequences, was ordered from Applied Biosystems, Inc. Taqman experiments were performed on the ABI 7500 Real-Time PCR System platform and each sample was run in quadruplicate. Ribosomal RNA (18S) was used as endogenous control to normalize quantification of each target within tissues using Applied Biosystems Sequence Detections Software version 1.3. The relative amount of viral mRNA (log 10) is presented in the final results.
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