Samples were treated with DNase I (New England Biolabs, Ipswich, MA, USA) to remove free DNA and with Proteinase K (Invitrogen, Waltham, MA, USA) to break down AAV capsids. Viral DNA was amplified using quantitative polymerase chain reaction (qPCR) with a set of primers and TaqMan probe (5′ 6‐FAM/ 3’ BHQ‐1, Millipore‐Sigma) targeting AAV inverted terminal repeats (ITRs) using the QuantStudio 7 PCR System (Applied Biosystems, Waltham, MA, USA). Purified linearized transgene plasmid was used as a standard to interpolate sample viral genome titers.
Taqman probe 5 6 fam 3 bhq 1
Manufactured by Merck Group
Sourced in United States
The TaqMan probe (5' 6‐FAM/ 3' BHQ‐1) is a fluorescent oligonucleotide probe used in real-time PCR (qPCR) assays. It consists of a fluorescent reporter dye (6‐FAM) at the 5' end and a quencher dye (BHQ‐1) at the 3' end. The probe binds to a specific target sequence and, during PCR amplification, the 5' nuclease activity of the DNA polymerase cleaves the probe, separating the reporter and quencher dyes, resulting in an increase in fluorescent signal.
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Lab products found in correlation
2 protocols using taqman probe 5 6 fam 3 bhq 1
1
Quantitative Assessment of AAV Titers
2
Determining AAV Viral Titer by TCID50
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AAV samples were serially diluted and co‐infected into HelaRC32 cells (ATCC, Manassas, VA, USA) with a human adenovirus 5 standard (ATCC, VR‐1516) in 96‐well plates. After 3 days of incubation at 37°C, DNA was extracted from cells and the virus replication endpoint was detected by qPCR measurement of the replicated viral genome. Median tissue culture infectious dose (TCID50) was calculated by the Spearman‐Kärber method. qPCR was conducted with a set of primers and TaqMan probe (5′ 6‐FAM/ 3’ BHQ‐1, Millipore‐Sigma) targeting AAV inverted terminal repeats (ITRs) using the QuantStudio 7 PCR System (Applied Biosystems).
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