Quantitative Analysis of Viral and Immune Markers

DNA was extracted from PBMC, BAL samples, and frozen lung tissue using the QIAamp DNA Mini Kit (QIAGEN). RNA extraction was performed by homogenizing pooled frozen lung biopsies (n = 3 per animal) in 1 mL TRIzol reagent (Invitrogen) using the MP Fastprep-24 (MP Biomedicals) and the RNeasy Mini Kit (QIAGEN). cDNA synthesis was performed using 5 μg total RNA and Superscript IV reverse transcriptase (Invitrogen) with oligo(dT) primers (Invitrogen). qPCR was performed in duplicate on an ABI Prism 7500 using TaqMan 2× Universal Master Mix (Applied Biosystems). DNA qPCR was performed with primers and probes specific for SVV ORF21 and the single copy gene OSM (70 (link)) (Supplemental Table 2). cDNA qPCR was performed with primers and probes specific for SVV ORF63 (17 (link)) (Supplemental Table 2), OSM (70 (link)), CXCL10 (Mf02788358_g1) (47 (link)), CDH5 (Mf00901470_m1), CLDN2 (Mf04369164_m1), CLDN10 (MF02862371_m1), CLDN18 (Mf02805373_m1), DDX58 (Mf02789247_m1), GAPDH (Mf04392546_g1), IFIT1 (Mf04355804_m1), IFN-α2 (Mf04256335_s1), IFN-γ (Mf02788577_m1), MX1 (Mf00895608_m1), and STAT1 (Hs01013996_m1) (Applied Biosystems).

Free full text: Click here

Ouwendijk W.J., van den Ham H.J., Delany M.W., van Kampen J.J., van Nierop G.P., Mehraban T., Zaaraoui-Boutahar F., van IJcken W.F., van den Brand J.M., de Vries R.D., Andeweg A.C, & Verjans G.M. (2020). Alveolar barrier disruption in varicella pneumonia is associated with neutrophil extracellular trap formation. JCI Insight, 5(21), e138900.

Publication 2020

QIAamp DNA Mini Kit TRIzol reagent MP Fastprep-24 RNeasy Mini Kit Superscript IV reverse transcriptase oligo(dT) primers ABI Prism 7500 TaqMan 2× Universal Master Mix GAPDH

Animal Biopsies Cdh5 Cdna Ddx58 Frozen Gapdh Gene Ifn γ Lung Oligo Primers Prism Reverse transcriptase Stat1 Synthesis Tissue Trizol

Corresponding Organization : Erasmus MC

Other organizations : ENPICOM (Netherlands), Utrecht University

Top 5 similar protocols

Variable analysis

independent variables

RNA extraction method (homogenizing pooled frozen lung biopsies in TRIzol reagent using MP Fastprep-24)
CDNA synthesis method (using Superscript IV reverse transcriptase with oligo(dT) primers)
QPCR method (performed in duplicate on an ABI Prism 7500 using TaqMan 2× Universal Master Mix)

dependent variables

Quantification of SVV ORF21 (DNA qPCR)
Quantification of SVV ORF63 (cDNA qPCR)
Quantification of OSM (DNA qPCR and cDNA qPCR)
Quantification of CXCL10 (cDNA qPCR)
Quantification of CDH5 (cDNA qPCR)
Quantification of CLDN2 (cDNA qPCR)
Quantification of CLDN10 (cDNA qPCR)
Quantification of CLDN18 (cDNA qPCR)
Quantification of DDX58 (cDNA qPCR)
Quantification of GAPDH (cDNA qPCR)
Quantification of IFIT1 (cDNA qPCR)
Quantification of IFN-α2 (cDNA qPCR)
Quantification of IFN-γ (cDNA qPCR)
Quantification of MX1 (cDNA qPCR)
Quantification of STAT1 (cDNA qPCR)

control variables

DNA and RNA extraction methods (QIAamp DNA Mini Kit and RNeasy Mini Kit)
Sample types (PBMC, BAL, and frozen lung tissue)

positive controls

None specified

negative controls

None specified

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!