Two to four curls from continuous FFPE tissue sections per specimen were collected in 1.5mL centrifuge tubes used for RNA isolation. The tubes containing the tissue curls were deparaffinized following the protocol for tube-sections in the Roche HighPure FFPET RNA Isolation spin-column kit (Catalog #06650775001) with the following modifications: maximum 10μM sections used were increased up to 4 curls rather than 1 per tube and d-limonene (histology grade) was used in place of xylene. The tissue sections were allowed to dry completely before proceeding to the RNA Isolation protocol. RNA isolation was performed using the same Roche HighPure FFPET RNA Isolation spin-column kit according to manufacturer’s specifications. RNA was quantified using the NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific), and a sample subset was additionally quality checked using the High Sensitivity RNA assay with the Qubit 2.0 Fluorometer (Invitrogen). All samples were additionally run using the High Sensitivity RNA Analysis Kit on a 5200 Fragment Analyzer Automated CE System (Agilent) to assess quality and determine nCounter assay input.
5200 fragment analyzer automated ce system
Manufactured by Agilent Technologies
Sourced in United States
The 5200 Fragment Analyzer Automated CE System is a laboratory instrument designed for the automated analysis of DNA, RNA, and protein samples. It utilizes capillary electrophoresis technology to separate and detect molecular fragments based on their size and charge. The system provides precise and reproducible results for a variety of applications, including gene expression analysis, DNA sequencing, and protein characterization.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (4 mentions)
Rneasy ffpe mini kit, by Qiagen (2 mentions)
Highpure ffpet rna isolation spin column kit, by Roche (1 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Nsolver software, by NanoString (1 mentions)
Canine io panel, by NanoString (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Rneasy ffpe kit, by Qiagen (1 mentions)
4 protocols using 5200 fragment analyzer automated ce system
1
FFPE Tissue RNA Isolation Protocol
2
Canine Immune-Oncology Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tumor biopsies were evaluated histologically to confirm that at least 50% of the sample contained tumor cells. Four 10-μm sections from each sample were used for RNA extraction with an RNeasy FFPE Mini kit (QIAGEN) then evaluated with RNA High Sensitivity assays on the Qubit 2.0 Fluorometer (Invitrogen/LifeTechnologies) and 5200 Fragment Analyzer Automated CE System (Agilent). NanoString (NanoString Technologies) gene expression analysis was performed using the NanoString Canine immune-oncology (IO) panel (24 (link)). NanoString data were collected using the nCounter Analysis System at the University of Arizona Genetics Core, software 4.0.1.8. Gene expression count data were preprocessed using nSolver software (NanoString) then normalized to housekeeping genes and batch effect regressed using panel standards. Differential gene expression analysis was completed using DESeq2 with corrections for multiple comparisons (25 (link)). For corrected P values, a threshold of 0.1 was used to determine statistical significance.
3
Canine Immuno-Oncology Gene Expression
4
Canine Immune Gene Expression Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted from archived frozen and FFPE tumor and normal tissues using Qiagen RNeasy and Qiagen RNeasy FFPE kits, respectively. For the FFPE samples, five to seven tissue sections (5-10 µm thick) were cut from each block and pooled for RNA extraction. RNA was extracted from flash-frozen lymph node tumor tissues using the RNeasy Plus Mini Kit (QIAGEN) following manufacturer protocol. Depending on starting material, samples were eluted in 30-50uL and were initially checked for quantity and purity on a Nanodrop ND-1000 Spectrophotometer (Thermo Fisher) prior to being stored at -80c until further processing. Samples were additionally quantity and quality checked using the RNA High Sensitivity assays on the Qubit 2.0 Fluorometer (Invitrogen/LifeTechnologies) and 5200 Fragment Analyzer Automated CE System (Agilent), respectively. NanoString gene expression analysis was performed using a custom-designed 48 gene canine immune panel derived from Rooney et al (37 (link)). The genes included in this panel are listed in Supplementary Table 1
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!