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INNOSC Theranostics and
Pharmacological Sciences Transcriptome-based RNA sequencing

with this array is cross-hybridization. Using full-length Human leukocyte antigen (HLA) genes and non-HLA
cDNAs, the mRNAs of paralogs can cross-hybridize to the genes are the genes and genetic loci that have been linked to the
wrong spots. A major challenge in analyzing microarray risk of RA. RNA-seq sheds insights into the causes of RA and
data is that it can be difficult to compare transcripts. 11 its therapy, making it easier to distinguish healthy individuals
from those with RA. Studies 27,28 comparing population
Another method is a sequencing-based technique or
RNA-seq. This technique largely replaces the microarrays, transcriptomes by analyzing the synovium, synovial cells,
white blood cells, and peripheral blood mononuclear cells
becoming popular for performing transcriptomics. The have been conducted. Studies have also revealed that patients
development of nanotechnology enables the sequencing of with RA have dysregulated levels of secretory phosphoprotein
hundreds of thousands of DNA molecules simultaneously. 1 (SPP1). Inflammation, T-cell activation, and apoptosis are
RNA sequencing is a process for determining the caused by SPP1. Proteoglycan 4 is detected in the severe stage
nucleotide sequence of an RNA molecule in a specific of RA. According to some studies, a correlation exists between
sample. The primary principle is based on a high-output the expression levels of transcripts linked to hematopoiesis,
sequencing technique with a computational technique interferon-mediated immunity, and cell trafficking and the
to capture and quantify transcripts present in an RNA advancement of arthritis. 27,28
extract. The nucleotide sequences generated are typically
approximately 100 bp in length, but they can range 3. Application of next-generation RNA
from 30 bp to more than 10,000 bp, depending on the sequencing (RNA-seq) in RA research
sequencing method used. In this method, RNA can first
be isolated from biological samples and converted into The cutting-edge method known as “next-generation RNA
double-stranded DNA molecules that serve as a template sequencing” is used to analyze transcriptomes and provides
for DNA polymerases during next-generation sequencing extremely high yields by determining the nucleotide order
(NGS) library preparation. These DNA copies are known in certain genomes or targeted DNA or RNA sections. The
as complementary DNA (cDNA), and the generation of discipline of RNA sequencing is revolutionized by NGS.
cDNA is initiated using the reverse transcriptase enzyme The three primary benefits of NGS are its cost-effectiveness,
and priming to form the first strand of cDNA. high throughput, and precise RNA characterization. The
current research using RNA-seq for RA theranostics is
The second strand is synthesized using DNA presented in Table 2.
polymerase, leading to the formation of double-stranded
cDNA. In the next step known as adapter ligation, the 3.1. Novel biomarker discovery
adapters ligated to the cDNA fragments and the resulting Biomarkers are biological substances found in bodily fluids
library molecules are amplified and sequenced. In this such as blood and tissues that indicate the presence of a
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method, short-read sequencing and map construction of disease. Some biomarkers for RA include rheumatoid factor,
the reads using a reference genome are performed using anticyclic citrullinated peptide, erythrocyte sedimentation
tools such as HISAT2 or STAR, and novel transcripts are rate, and C-reactive protein. Imaging biomarkers such as
assembled using tools such as StringTie. Other tools such X-rays and MRIs can detect joint erosion and synovitis.
as HTseq help in estimating the expression levels of the Genes such as SNPs and HLA-DRB1 can also function as
transcripts. Three factors, namely transcript level, transcript biomarkers, helping to determine the severity and course
length, and sequencing depth, affect the number of read of a disease. Elevated levels of chemokines and cytokines
transcripts that can be detected in a sample. RNA-seq during the proinflammatory phase can also be detected in
does not require prior genomic information, which makes certain patients with RA. 29-32
it suitable for analyzing transcriptomes in non-model
species. 13,14 Transcriptome analysis is generally performed 3.2. Identification of dysregulated pathways
for two major purposes; one is qualitative estimation, RNA sequencing helps in determining the RA pathways.
that is, identification of genes and their isoforms, and the RNA-seq facilitates the determination of transcriptome
other is quantitative analysis, that is, estimating the gene abundance levels and allows for the comparison of gene
expression or relative expression in different biological expression patterns with those of a healthy individual.
samples. RNA is less stable than DNA; however, the The genes that are dysregulated help in indicating the
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transcriptome is considerably more cell- and tissue-specific molecular pathways. 23
than the genome and is crucial for the generation of high-
quality data. Table 1 shows the transcriptomes used for 3.3. Gene ontology
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RNA-seq to demonstrate the relevance of the approach Gene ontology helps in the processing of RNA-seq raw
across several conditions beyond RA. data. It involves quality control to remove any reading of

Volume 8 Issue 1 (2025) 17 doi: 10.36922/itps.4449