Quantification of RNA and Protein Levels

Total RNA was extracted using TRIzol reagent (Invitrogen) and quantified by NanoDrop-100 spectrophotometer. Real-time quantitative PCR was performed with an iQ5 (Bio-Rad Laboratories) [8 (link)]. TaqMan quantitative PCR assays (Applied Biosystems, Life Technologies) were run using primers/probes: TNF (Mm00443258 or Hs01113624), ADAM10 (Mm00545742 or Hs00153853), ADAM17 (Mm00456428 or Hs01041915), 18s (Mm03928990), and GAPDH (Hs02758991). Fold variation analyzed using delta-delta Ct (∆∆Ct) formula [18 (link)].
Proteins were assessed by western blot [8 (link)] using rabbit-anti-β-actin peroxidase (Sigma-Aldrich) or anti-ADAM17 (Abcam) followed by HRP-anti-rabbit IgG and detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).

Free full text: Click here

Cooley L.F., Martin R.K., Zellner H.B., Irani A.M., Uram-Tuculescu C., El Shikh M.E, & Conrad D.H. (2015). Increased B Cell ADAM10 in Allergic Patients and Th2 Prone Mice. PLoS ONE, 10(5), e0124331.

Publication 2015

TRIzol reagent anti-ADAM17 SuperSignal West Pico Chemiluminescent Substrate

Actin Adam17 Anti igg Assays Gapdh Peroxidase Primers Proteins Rabbit Real time pcr Trizol Western blot

Corresponding Organization : Queen Mary University of London

Top 5 similar protocols

Variable analysis

independent variables

None explicitly mentioned

dependent variables

MRNA expression levels of TNF, ADAM10, and ADAM17
Protein levels of ADAM17

control variables

RNA extraction method (TRIzol reagent)
RNA quantification method (NanoDrop-100 spectrophotometer)
Real-time PCR platform (iQ5, Bio-Rad Laboratories)
Real-time PCR assay (TaqMan, Applied Biosystems, Life Technologies)
Reference genes (18s, GAPDH)
Western blot method (rabbit-anti-β-actin peroxidase, anti-ADAM17 antibody, HRP-anti-rabbit IgG, SuperSignal West Pico Chemiluminescent Substrate)

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

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?

Revolutionizing how scientists
search and build protocols!