Quantifying Pro-Inflammatory Cytokine Expression

RNA was extracted from cartilage explants and synoviocytes as previously described [33 (link)]. Total RNA concentration was determined using a Nanodrop-2000c (Thermo Fisher Scientific). Complimentary DNA (cDNA) was prepared [33 (link)]. Real-time qPCR was used to measure relative gene expression for markers of three pro-inflammatory cytokines: Interleukin 1 beta (IL1β), Interleukin 6 (IL6) and Tumour Necrosis Factor alpha (TNFα) compared to the reference gene glyceraldehydes-3-phosphatedhyrogenase (GAPDH) as described previously using SYBR Green technology [33 (link)]. Primer sequences are listed in Table 2.Table 2

Primer sequences of the reference gene (GAPDH) and pro-inflammatory cytokines IL1β, IL6 and TNFα used in the study

Gene	GeneBank Accession	Direction	Primer Sequence (5′-3′)
GAPDH	NM_001163856	Forward	GCATCGTGGAGGGACTCA
GAPDH	NM_001163856	Reverse	GCCACATCTTCCCAGAGG
IL-1β	NM_001317261	Forward	GCCTAAGAATACTACATCCAGAGA
IL-1β	NM_001317261	Reverse	GGCATTGATTAGACAACAGTGAA
IL-6	NM_001082496	Forward	CTGCTCCTCGTGATGGCTAC
IL-6	NM_001082496	Reverse	CCGAGGATGTACTTAATGTGCTG
TNF-α	NM_001081819	Forward	CCTTCCACTCAATCAACCCTCT
TNF-α	NM_001081819	Reverse	CACGCCCACTCAGCCACT

Primer sequences of the reference gene (GAPDH) and proinflammatory cytokines IL1β, IL6 and TNFα used in the study

Free full text: Click here

Rubio-Martínez L.M., Rioja E., Castro Martins M., Wipawee S., Clegg P, & Peffers M.J. (2017). Local anaesthetics or their combination with morphine and/or magnesium sulphate are toxic for equine chondrocytes and synoviocytes in vitro. BMC Veterinary Research, 13, 318.

Publication 2017

Nanodrop-2000c

Cartilage Cdna Cytokines Gapdh Gene Gene expression Il1β Inflammatory Interleukin 6 Primer Sybr green Synoviocytes Tnfα

Corresponding Organization : University of Liverpool

Other organizations : Rajamangala University of Technology Srivijaya

Top 5 similar protocols

Variable analysis

independent variables

None explicitly mentioned

dependent variables

Relative gene expression for markers of three pro-inflammatory cytokines: Interleukin 1 beta (IL1β), Interleukin 6 (IL6) and Tumour Necrosis Factor alpha (TNFα)

control variables

Reference gene glyceraldehydes-3-phosphatedhyrogenase (GAPDH)

controls

Positive control: GAPDH (reference gene)
Negative control: 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!