Extracta DNA Prep for PCR is a two-component reagent kit for rapid extraction of PCR-ready genomic DNA from a variety of tissues. Samples are processed in less than 30 minutes with minimal hands-on time and technical skill. Extracted genomic DNA is suitable for sensitive downstream PCR applications including end-point PCR, High Resolution Melt Analysis (HRM) and quantitative real-time PCR (qPCR) without requiring any additional clean-up. In addition, the extracted DNA may be
used in multiplexed PCR applications such as transgene or knock-out analyses. Tissue extractions can be done in tubes, plates or deep-well blocks to allow for adaptation to workflow and automation on liquid-handling workstations.
2 mm tail snips were extracted in 150 µL of Extracta DNA Prep for PCR – Tissue or reagents from competitor products according to each manufacturers instructions. 1.5 µL of each extract was used in 20 µL PCR reactions of mouse brain-derived neurotropic factor using PerfeCTa SYBR Green FastMix. PCR data was obtained in triplicate and converted to number of copies of target detected using a standard curve generated from purified mouse genomic DNA.
Comparison of Extract Yield: Mouse Ears
1 mm diameter mouse ear punches were extracted in 100 µL of Extracta DNA Prep for PCR – Tissue or reagents from competitor products according to each manufacturers instructions. 5.0 µL of each extract was used in 20 µL PCR reactions of mouse brain-derived neurotropic factor using PerfeCTa SYBR Green FastMix. PCR data was obtained in triplicate and converted to number of copies of target detected using a standard curve generated from purified mouse genomic DNA.
Extracta reduces time to results with a rapid, convenient protocol.
Disease-associated microglia (DAMs), that surround beta-amyloid plaques, represent a transcriptionally-distinct microglial profile in Alzheimer’s disease (AD). Activation of DAMs is dependent on triggering receptor expressed on myeloid cells 2 (TREM2) in mouse models and the AD TREM2-R47H risk variant reduces microglial activation and plaque association in human carriers. Interestingly, TREM2 has also been identified as a microglial lipid-sensor, and recent data indicates lipid droplet accumulation in aged microglia, that is in turn associated with a dysfunctional proinflammatory phenotype. However, whether lipid droplets (LDs) are present in human microglia in AD and how the R47H mutation affects this remains unknown.
To determine the impact of the TREM2 R47H mutation on human microglial function in vivo, we transplanted wild-type and isogenic TREM2-R47H iPSC-derived microglial progenitors into our recently developed chimeric Alzheimer mouse model. At 7 months of age scRNA-seq and histological analyses were performed.
Here we report that the transcriptome of human wild-type TREM2 and isogenic TREM2-R47H DAM xenografted microglia (xMGs), isolated from chimeric AD mice, closely resembles that of human atherosclerotic foam cells. In addition, much like foam cells, plaque-bound xMGs are highly enriched in lipid droplets. Somewhat surprisingly and in contrast to a recent in vitro study, TREM2-R47H mutant xMGs exhibit an overall reduction in the accumulation of lipid droplets in vivo. Notably, TREM2-R47H xMGs also show overall reduced reactivity to plaques, including diminished plaque-proximity, reduced CD9 expression, and lower secretion of plaque-associated APOE.
Altogether, these results indicate lipid droplet accumulation occurs in human DAM xMGs in AD, but is reduced in TREM2-R47H DAM xMGs, as it occurs secondary to TREM2-mediated changes in plaque proximity and reactivity.
In the absence of vaccines or drugs, insecticides are the mainstay of Aedes-borne disease control. Their utility is challenged by the slow deployment of resources, poor community compliance and inadequate household coverage. Novel application methods are required.
Methodology and principal findings
A 10% w/w metofluthrin “emanator” that passively disseminates insecticide from an impregnated net was evaluated in a randomized trial of 200 houses in Mexico. The devices were introduced at a rate of 1 per room and replaced at 3-week intervals. During each of 7 consecutive deployment cycles, indoor resting mosquitoes were sampled using aspirator collections. Assessments of mosquito landing behaviours were made in a subset of houses. Pre-treatment, there were no differences in Aedes aegypti indices between houses recruited to the control and treatment arms. Immediately after metofluthrin deployment, the entomological indices between the trial arms diverged. Averaged across the trial, there were significant reductions in Abundance Rate Ratios for total Ae. aegypti, female abundance and females that contained blood meals (2.5, 2.4 and 2.3-times fewer mosquitoes respectively; P<0.001). Average efficacy was 60.2% for total adults, 58.3% for females, and 57.2% for blood-fed females. The emanators also reduced mosquito landings by 90% from 12.5 to 1.2 per 10-minute sampling period (P<0.05). Homozygous forms of the pyrethroid resistant kdr alleles V410L, V1016L and F1534C were common in the target mosquito population; found in 39%, 24% and 95% of mosquitoes collected during the trial.
This is the first randomized control trial to evaluate the entomological impact of any volatile pyrethroid on urban Ae. aegypti. It demonstrates that volatile pyrethroids can have a sustained impact on Ae. aegypti population densities and human-vector contact indoors. These effects occur despite the presence of pyrethroid-resistant alleles in the target population. Formulations like these may have considerable utility for public health vector control responses.
Insecticide resistance across sub-Saharan Africa may impact the continued effectiveness of malaria vector control. We investigated the association between carbamate and pyrethroid resistance with Anopheles gambiae s.l. parity, Plasmodium falciparum infection, and molecular insecticide resistance mechanisms in Guinea. Pyrethroid resistance was intense, with field populations surviving ten times the insecticidal concentration required to kill susceptible individuals. The L1014F kdr-N1575Y haplotype and I1527T mutation were significantly associated with mosquito survival following permethrin exposure (Prevalence Ratio; PR = 1.92, CI = 1.09–3.37 and PR = 2.80, CI = 1.03–7.64, respectively). Partial restoration of pyrethroid susceptibility following synergist pre-exposure suggests a role for mixed-function oxidases. Carbamate resistance was lower and significantly associated with the G119S Ace-1 mutation. Oocyst rates were 6.8% and 4.2% among resistant and susceptible mosquitoes, respectively; survivors of bendiocarb exposure were significantly more likely to be infected. Pyrethroid resistant mosquitoes had significantly lower parity rates than their susceptible counterparts (PR = 1.15, CI = 1.10–1.21). Our findings emphasize the need for additional studies directly assessing the influence of insecticide resistance on mosquito fitness.
Extracta DNA Prep for PCR
"I needed to extract DNA from fungal tissue for one of our teaching labs. It worked amazing for further PCR reactions."
Andres P. | University Lab Coordinator
Extracta DNA Prep for PCR - Tissue
"Extracta DNA Prep for PCR is one of the best, fastest and easiest to use reagent for genomic DNA isolation from human cells."