Long-probe quantitative amplifiedsignal (LQAS) assay master
LQAS is often used as a way to do qPCR that is methylation-specific besides methylation-specific qPCR.
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Duplex recovery is assessed by the final number of dsDNA bases sequenced (in Gb)and total dsDNA coverage (in genome equivalents) across total bases sequenced
Basically, "duplex recovery" is calculated as: identify every dsDNA read (a read which has at least 1 read from the opposite strand); recovery is "the number of bases on all dsDNA reads divided by the number of bases on all reads". Max is 100%.
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- Aug 2025
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Quantitative real-time PCR (RT-qPCR) was used for quantification ofcfRNA. An RNA-specific primer was designed to cover a 97-bp ampli-con spanning 2 exon boundaries in the housekeeping gene GAPDH(forward 5′-GATCATCAGCAATGCCTCCT-3′, reverse 5′-TGTGGTCATGAGTCCTTCCA-3′). A DNA-specific primer was designed to covera 78-bp transcriptionally silent region of chromosome 12 (forward5′-TACGGTTGGTCCTTTCTTCG-3′, reverse 5′-TTTCCTTTGGGTCTGAATGC-3′). Reverse transcription was first performed using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). qPCRwas then run using 2X Power SYBR Green PCR Master Mix (ThermoFisher Scientific) on an Applied Biosystems 7500 Fast Real-Time PCRor QuantStudio 7 Pro instruments. Universal Human Reference RNA(Thermo Fisher Scientific) was run in parallel to generate a standardcurve, and cfRNA concentrations were calculated by comparing theRNA-specific Ct value of the sample to the standard curve.
They quant cfRNA after extraction but before library prep & capture. They use RT-qPCR of a GAPDH region vs a standard curve made using Universal Human Reference RNA. Therefore their actual RNA quant might be off - it's more like a rough overall expression estimate.
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