Total RNA was extracted from 50 µl plasma using the EZNA Total RNA Kit I (Omega Bio-Tek), eluting in 100 µl water. 10 µl of RNA was added into a 1× 1-step TaqMan Reverse Transcriptase PCR reaction mix (Applied Biosystems) that included 10 pmol of F-MLV-specific primers: F-MLV sense, 5’-GGACAGAAACTACCGCCCTG-3’; F-MLV antisense, 5’-ACAA- CCTCAGACAACGAAGTAAGA-3’; and F-MLV probe, FAM-TCGCCACCCAGCAGTTTCAGCAGC-TAMRA. Quantitative PCR (qPCR) conditions in a Bio-Rad CFX96 cycler were as follows: 48°C for 15 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, 60°C for 1 min. In vitro transcribed F-MLV-RNA of known copy number was used as a standard.
Taqman reverse transcriptase pcr reaction mix
Manufactured by Thermo Fisher Scientific
The TaqMan Reverse Transcriptase PCR reaction mix is a reagent designed for reverse transcription and real-time PCR amplification of RNA targets. It contains a reverse transcriptase enzyme, DNA polymerase, and necessary buffers and reagents required for the two-step process of reverse transcription and subsequent PCR amplification.
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Lab products found in correlation
2 protocols using taqman reverse transcriptase pcr reaction mix
1
Quantitative PCR-based F-MLV RNA detection
2
Quantitative PCR for Plasma Viral Load
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Plasma viral load was measured by quantitative real-time PCR (qPCR)64 (link). RNA was extracted from 50 μl plasma using the Qiagen RNAeasy kit and eluted in 100 μl water. RNA (10 μl) was added to 1 × 1-step TaqMan Reverse Transcriptase PCR reaction mix (Applied Biosystems) along with 10 pmol of the following primers and probe: F-MuLV sense, 5′-GGACAGAAACTACCGCCCTG-3′; F-MuLV antisense, 5′-ACAA- CCTCAGACAACGAAGTAAGA-3′; and F-MuLV probe, FAM-TCGCCACCCAGCAGTTTCAGCAGC-TAMRA. Real-time PCR was performed in a Bio-Rad CFX96 cycler using these thermocycling conditions: 48 °C for 15 min, 95 °C for 10 min, 40 cycles of 95 °C for 15 s, 60 °C for 1 min. T7-transcribed F-MuLV RNA standards were used to interpolate absolute F-MuLV RNA copy numbers in the plasma.
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