Staged young adult tau transgenic C. elegans were grown from eggs at 20 °C for 3 days on 5XPEP plates, washed off plates in M9 buffer, and collected by centrifugation. RNA was extracted from ~100 μL of packed worms per sample using Tri-Reagent (Molecular Research Center) as per manufacturer instructions. Poly(A) tail lengths were assayed using the USB Poly(A) Tail-Length Assay Kit (Affymetrix) as per manufacturer instructions with the following modification to the PCR amplification step. The PCR reaction consisted of 10 μL of HotStart Taq, 7 μL of nuclease-free water, 0.5 μL of 10 μM gene-specific primer (see below), 0.5 μL of 10 μM Universal PCR Reverse Primer, and 2 μL of the diluted reverse transcription sample. A three-step PCR reaction with 40 cycles of amplification was used to amplify the signal. PCR products were run on a QIAexcel Advanced capillary electrophoresis instrument (Qiagen) using a QIAxcel DNA high resolution Kit with QX DNA Size Marker pUC18/HaeIII (catalog #929550) at 20 ng/uL and QX alignment marker 15 bp/600 bp (catalog #929530). Samples were processed using the 0M400 method with a 20 s sample injection time and analyzed with the QIAxcel ScreenGel Software for major peak, median signal, and concentration.
Qx alignment marker 15 bp 600 bp
Manufactured by Qiagen
The QX Alignment Marker 15 bp/600 bp is a molecular weight marker used for the size estimation of DNA fragments in gel electrophoresis. It contains DNA fragments of defined sizes (15 base pairs and 600 base pairs) that serve as reference points for determining the size of unknown DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Usb poly a tail length assay kit, by Thermo Fisher Scientific (2 mentions)
Qiaexcel advanced capillary electrophoresis instrument, by Qiagen (2 mentions)
Qiaxcel dna high resolution kit with qx dna size marker puc18 haeiii, by Qiagen (2 mentions)
Tri reagent, by Molecular Research Center (2 mentions)
Qx dna size marker 25 500 bp, by Qiagen (1 mentions)
Qbase software, by CellCarta (1 mentions)
Prism 6, by GraphPad (1 mentions)
3 protocols using qx alignment marker 15 bp 600 bp
1
Poly(A) Tail Length Profiling in C. elegans
2
Quantitative Expression Analysis of AQP4 in Mice
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Analysis of relative mRNA expression was performed using qbase + software (Biogazelle, Zwijnaarde, Belgium), with relative abundance (RQ values) calculated using a series of normalization methods based on the classical delta-delta Ct method and MIQE—compliant procedures [24 (link)]. The RT-qPCR cycle threshold (Ct) values were the input data in the qbase + program. Results were calculated for 100% PCR efficiency and ‘unpaired’ experimental design.
Statistical analysis for mouse AQP4 expression was performed with GraphPad Prism 6.07 (GraphPad Software, San Diego, USA) by processing the qbase + RQ values using an unpaired t test. P values < 0.05 were considered statistically significant.
After the amplification, PCR products were analyzed by high-resolution capillary electrophoresis with the Qiaxcel DNA High Resolution Kit, QX Alignment Marker 15 bp/600 bp and the QX DNA Size Marker 25–500 bp at a concentration of 30 ng/µl was used (Qiagen). The separation was performed using the OM800 method of the Qiaxcel System with the following parameters: 4 kV and 5 s for alignment marker injection, 5 kV and 10 s for the sample injection and 3 kV for 800 s for separation. The results were displayed as gel images as obtained from QIAxcel system software.
Statistical analysis for mouse AQP4 expression was performed with GraphPad Prism 6.07 (GraphPad Software, San Diego, USA) by processing the qbase + RQ values using an unpaired t test. P values < 0.05 were considered statistically significant.
After the amplification, PCR products were analyzed by high-resolution capillary electrophoresis with the Qiaxcel DNA High Resolution Kit, QX Alignment Marker 15 bp/600 bp and the QX DNA Size Marker 25–500 bp at a concentration of 30 ng/µl was used (Qiagen). The separation was performed using the OM800 method of the Qiaxcel System with the following parameters: 4 kV and 5 s for alignment marker injection, 5 kV and 10 s for the sample injection and 3 kV for 800 s for separation. The results were displayed as gel images as obtained from QIAxcel system software.
3
Poly(A) Tail Length Profiling in C. elegans
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