Each tissue was transferred to a liquid nitrogen-precooled mortar and ground into a powder using a pestle. Then total RNA was extracted from the tissue specimens and NPC cell lines using the TRIzol reagent (Invitrogen), according to the manufacturer's instructions. Approximately 1 μg total RNA was reverse-transcribed using the ThermoScript reverse transcription-Polymerase Chain Reaction (RT-PCR) system (Invitrogen,), based on the manufacturer's instructions.
Thermoscript reverse transcription polymerase chain reaction rt pcr system
Manufactured by Thermo Fisher Scientific
The ThermoScript reverse transcription polymerase chain reaction (RT-PCR) System is a laboratory instrument designed for the amplification and detection of RNA sequences. It performs the process of reverse transcription, where RNA is converted into complementary DNA (cDNA), followed by the polymerase chain reaction (PCR) to amplify the target DNA sequence.
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3 protocols using thermoscript reverse transcription polymerase chain reaction rt pcr system
1
Total RNA Extraction and Reverse Transcription
2
Quantitative RT-PCR for mRNA Expression
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Total RNA was extracted by mirVana PARIS kit (Ambion; Thermo Fisher Scientific, Waltham, MA) following the manufacturer's instructions. Five micrograms of RNA were used for reverse transcription by ThermoScript reverse transcription polymerase chain reaction (RT-PCR) System (Invitrogen; Thermo Fisher Scientific) yielding complementary DNA template. Quantitative real-time PCR amplification and data analysis were performed using 7500 Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific). Taq Man gene expression assays were used to quantify messenger RNA (mRNA) expression of the respective genes. mRNA expression levels of each sample were normalized to 18S RNA (TaqMan RNA control reagents; Applied Biosystems). All mRNA expression data were calculated as fold change from naı ¨ve animal tissues.
3
Quantitative RT-PCR for Gene Expression
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Total RNA was extracted by mirVana Paris kit (Ambion, Thermo Fisher Scientific, Waltham, MA) following the manufacturer's instructions. Five micrograms of RNA were used for reverse transcription by ThermoScript reverse transcription polymerase chain reaction (RT-PCR) System (Invitrogen, Thermo Fisher Scientific) yielding cDNA template. Quantitative real-time PCR amplification and data analysis were performed using 7500 Fast Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific). Taq Man gene expression assays were used to quantify mRNA expression of the respective genes. mRNA expression levels of each sample were normalized to 18S RNA (Taq Man rRNA control reagents; Applied Biosystems, Thermo Fisher Scientific). All mRNA expression data was calculated as fold change from naïve animal tissues.
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