21 Matching Annotations
  1. Jan 2021
    1. Indeed, the cell-lysate approach was found to be associated with superior sensitivity in some cases5.
    2. using a commercially available reagent (Bio-Rad SPR) that generates RT-qPCR-ready cell lysates with minimal manipulations. Similar reagents are now available from a number of sources (Ambion Cells-to-CT, Invitrogen CellsDirect, Roche RealTime Ready Cell Lysis, etc.)
    1. we demonstrate improved PCR-enhancing cocktails containing nonionic detergent, l-carnitine, d-(+)-trehalose, and heparin. These cocktails, in combination with two inhibitor-resistant Taq mutants, OmniTaq and Omni Klentaq, enabled efficient amplification of exogenous, endogenous, and high-GC content DNA targets directly from crude samples containing human plasma, serum, and whole blood without DNA purification.
    1. We found that a lysis solution containing both the non-ionic detergent (IGEPAL CA-630, chemically equivalent to Nonidet P-40 or NP-40) and bovine serum albumin (BSA) gave the best RT-qPCR yield. This direct lysis to reverse transcription protocol outperformed a column-based extraction method using a commercial kit.
  2. Dec 2020
  3. Nov 2020
    1. Copy numbers of the oriV region and gfp gene on the plasmid relative to a chromosomal gene were determined by qPCR. The chromosomal gene dapA was set up as the reference gene
    1. Designed probes were modified at the 5′ end with the fluorophore FAM for the reference gene cysG and HEX for the plasmid marker oriT, and furthermore modified at the 3′ end with the quencher BHQ-1

      plasmid copy number qPCR assay.

      Why cysG: Siroheme synthase gene?

  4. Jun 2020
    1. the lowest copy number sample points impacted faster and sronger, then you just need to stabilize by adding a neutral nucleic acid background in your standard curve. I usually use water containig 10ng/microl yeast tRNA to perform serial dilutions. This will first create a reaction background similar to your RTQPCR reaction, but also stabilize your DNA copies; I can freeze and thaw (min 20C) more than 50 times the same standard curve sample without any loss in Cts, from 10E6 to 10E2 copies. When I tested the same standard curve but diluted in water only, then the 10E2 started to be slightly affected after one freeze and thaw and then crashed further; then higher copy numbers samples were also affected after 2 to 3 freeze and thaw.
  5. www.gene-quantification.de www.gene-quantification.de
    1. t is important that the threshold is set at a fixed level for all samples that are to be compared
    1. A known copy (104/reaction) of Oncorhynchus keta (O. keta) was added in the DNAse and RNAse free water and the Cq value obtained acted as a reference point. If the Cq value of a wastewater sample increases compared to the reference Cq value, the sample is considered to have PCR inhibitors.

      Was this RNA or DNA that was spiked in?

      • O. Keta is a salmon fish
      • The reference cited here (Haugland et al., 2005) uses a DNA standard of O.keta 16s rRNA gene
  6. May 2020
    1. The Rn value, or normalized reporter value, is the fluorescent signal from SYBR Green normalized to (divided by) the signal of the passive reference dye for a given reaction. The delta Rn value is the Rn value of an experimental reaction minus the Rn value of the baseline signal generated by the instrument. This parameter reliably calculates the magnitude of the specific signal generated from a given set of PCR conditions
    1. Although unnecessary for simple singleplex amplifications, spectral calibration is critical for multiplexed assays so that overlapping fluorescent signals can be resolved from one another.
  7. Apr 2020
    1. a defined quantity of a nonhuman control virus (seal herpes virus [SHV], kindly provided by H. G. M. Niesters, University of Rotterdam, The Netherlands) was spiked into each sample prior to DNA and RNA extraction. Since constant DNA quantities of the control virus are coextracted even when RNA isolation kits are used (H. G. M. Niesters, personal communication; our own unpublished observations), the virus can also serve as a control in RNA virus detection assays.
    1. We investigated the biodistribution of SARS-CoV-2 among different tissues of inpatients with coronavirus disease 2019 (COVID-19)

      It would have been good to include 2-3 negative control samples in the data

  8. Jul 2019
  9. Apr 2019
    1. organisms require physical lysis mainly based on heat,(31) pressure,(32) sonication with sound waves,(33) and bead milling.(34)
  10. Mar 2019
  11. www.microbiologyresearch.org www.microbiologyresearch.org
    1. The fluorescence signal atCT37±7 corre-sponds to the no-template-control andrepresents bacterial DNA contamination inthe commercially supplied reagents.