The serum samples were stored at −80°C until further processing. DNA was extracted using the QIAamp DNA Mini kit (Qiagen) according to the manufacturer's protocol. Serum samples (200 μl) were used for DNA extraction after the addition of 10 μg of poly(A) (Roche Diagnostics K. K.) as a carrier RNA. A volume of 50 μl of the final eluate was used. Extracted DNA was stored at −20°C until further use.
Poly a
Manufactured by Roche
Sourced in Germany
Poly(A) is a laboratory equipment used in molecular biology and genetics research. It is designed to isolate and purify polyadenylated RNA (messenger RNA) from biological samples. The core function of Poly(A) is to facilitate the extraction and separation of mRNA molecules from total RNA extracts, which is an essential step in various gene expression analysis techniques.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Binding buffer, by Roche (1 mentions)
Rnalater stabilization solution, by Thermo Fisher Scientific (1 mentions)
High pure viral nucleic acid kit, by Roche (1 mentions)
Heparin, by Merck Group (1 mentions)
Polyu, by Merck Group (1 mentions)
4 protocols using poly a
1
DNA Extraction from Serum Samples
2
Murine Tissue and Blood Sampling for RNA Analysis
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Three mice of each group were euthanized and perfused with 50 ml of cold phosphate-buffered saline through the left ventricle of the heart. Then, the tissues of the mesenteric lymph node, axillary lymph node, brain, lung, liver, spleen, kidney, stomach, and intestine were collected from days 1–3 and submerged in RNAlater Stabilization Solution (Thermo Fisher Scientific K.K.) for storage. The samples were stored at -80 °C for 2–4 days until RNA extraction. Total RNA was extracted from the collected tissues using TRIzol reagent (Thermo Fisher Scientific K.K.) according to the manufacturer's protocol.
Ten mice from each group, which were used for the hematology and serum biochemistry analyses described below, were euthanized. Whole blood was collected from days 1–3. 20 μL of the collected blood was immediately mixed with binding buffer (Roche Applied Science, Penzberg, Germany) containing poly [A] (Roche Applied Science) and phosphate-buffered saline and stored at -80 °C until RNA extraction. Total RNA was extracted using High Pure Viral Nucleic Acid Kit (Roche Applied Science) according to the manufacturer's protocol.
Ten mice from each group, which were used for the hematology and serum biochemistry analyses described below, were euthanized. Whole blood was collected from days 1–3. 20 μL of the collected blood was immediately mixed with binding buffer (Roche Applied Science, Penzberg, Germany) containing poly [A] (Roche Applied Science) and phosphate-buffered saline and stored at -80 °C until RNA extraction. Total RNA was extracted using High Pure Viral Nucleic Acid Kit (Roche Applied Science) according to the manufacturer's protocol.
3
Polyadenylic Acid Carrier DNA in Proteinase K Digestion
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4
Tau Protein Aggregation Kinetics
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The tau protein was incubated at 20 μM in 384-well low-volume
microplate with 20 μM ThT. The RNA polyC (Sigma P4903), polyA
(Roche 10108626001) or polyU(Sigma P9528) were added at 200 μM.
Heparin (Sigma H6279) was added at a concentration of 5 μM in
supplementary experiments (Figure S11 ).
The working volume was 20 μL. The fluorescence was bottom read
in a BMG fluoroStar Omega instrument with excitation and emission
wavelengths of 440 and 480 nm, respectively. Each condition was prepared
independently in three or two different wells.
ThT kinetic curves
were first normalized between 0 and 1. They were then fitted using
the curve_fit python function with the following
equation: where F represents the final
fluorescent intensity, k represents the growth rate,
and t1/2 represents the aggregation halftime.
Each replicate was fitted independently. The presented error bars
on the aggregation halftimes represent the standard deviation over
the output t1/2 obtained from the fit
of each replicate. The fitting functions are shown inFigure S3 . tau187-WT signal was normalized with
the maximum intensity of tau187-P301L for visualization inFigure 2 A, since it did not
increase.
microplate with 20 μM ThT. The RNA polyC (Sigma P4903), polyA
(Roche 10108626001) or polyU(Sigma P9528) were added at 200 μM.
Heparin (Sigma H6279) was added at a concentration of 5 μM in
supplementary experiments (
The working volume was 20 μL. The fluorescence was bottom read
in a BMG fluoroStar Omega instrument with excitation and emission
wavelengths of 440 and 480 nm, respectively. Each condition was prepared
independently in three or two different wells.
ThT kinetic curves
were first normalized between 0 and 1. They were then fitted using
the curve_fit python function with the following
equation: where F represents the final
fluorescent intensity, k represents the growth rate,
and t1/2 represents the aggregation halftime.
Each replicate was fitted independently. The presented error bars
on the aggregation halftimes represent the standard deviation over
the output t1/2 obtained from the fit
of each replicate. The fitting functions are shown in
the maximum intensity of tau187-P301L for visualization in
increase.
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