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INNOSC Theranostics and
Pharmacological Sciences Transcriptome-based RNA sequencing

as matrix metalloproteinase (MMP)-9 and MMP-3 for cryopreserving the synovial tissue that minimizes
are secreted. Moreover, enhanced proliferation and batch effects and permits further digestion. To avoid
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reduced responsiveness to apoptotic signals are observed. any contaminated proteins or DNA, the extracted RNA is
Consequently, the synovium develops an aggressive nature, purified. Reverse transcriptase is used to convert RNA into
resulting in inflammation and hyperplasia, ultimately cDNA. Polymerase chain reaction (PCR) is used to amplify
causing discomfort and loss of function. However, over the the fragmented cDNA library to provide sufficient material
past 10 years, several fibroblast subpopulations have been for sequencing. An Illumina NovaSeq or HiSeq sequencing
identified through transcriptomic technologies and single- platform is used to load the prepared library.
cell sequencing. Low-quality reads and adaptor sequences are eliminated
In each previously reported experiment, the synovial from the raw sequencing reads through processing.
tissue was collected from limbs that had been amputated or The abundance of each transcript is measured using
from victims’ limbs within 6 h of their death. Studies often bioinformatics techniques (gene expression analysis).
end at one of two resolution levels: whole-tissue samples or Functional analysis of the DEGs is performed to gain
individual cellular populations. 42,43 Two primary methods insights into their biological functions and possible roles in
are commonly used for the isolation and investigation of the pathophysiology of RA. Researchers can use RNA-seq
synovial cells, also known as synovial fibroblasts: the explant to obtain useful information on the gene expression profile
outgrowth approach and the enzymatic digestion approach. of synovial cells in RA by following the techniques depicted
The enzymatic digestion strategy focuses on breaking down in Figure 3. Using these data, we can better understand the
the extracellular matrix and disrupting cell adhesion to this mechanisms underlying RA and establish new diagnostic
matrix to generate a suspension of synthetic cells. However, and treatment targets.
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a range of enzymes, concentrations, and digestion durations 5. Identification of DEGs associated with RA
can be used to achieve this. Owing to the straightforward
nature of the explant outgrowth approach, it is reported pathogenesis
more frequently. When small segments of synovial tissue The most common type of autoimmune inflammatory
are cultivated, adherent cells can move out of the tissue arthritis in adults is RA. In addition to increasing the
and begin to proliferate. 42,44 After 7 days, the remaining mortality rate, RA significantly reduces the quality of life
tissues should be removed to cultivate cells. Although in terms of health and impairs one’s ability to perform
the explant outgrowth approach is easier to use than the everyday chores, including domestic and job-related
enzymatic digestion approach, it has certain limitations. responsibilities. 37,41 Five of every 1000 people have RA,
Favoring synovial populations that proliferate rapidly and which certainly causes serious damage to joints and
are mobile enough to escape the tissue while ignoring other disability. Joint synovitis, which can appear as exudation,
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populations is risky. Furthermore, there exists a technique synovitis, or other diseases in the joint cavity, is a

Figure 3. Transcriptomic gene expression profiling of synovial cells in rheumatoid arthritis

Volume 8 Issue 1 (2025) 24 doi: 10.36922/itps.4449