NGS/Sequencing

Miller et al., 2022 “A preliminary, observational study using whole-blood RNA sequencing reveals differential expression of inflammatory and bone markers post-implantation of percutaneous osseointegrated prostheses“: PLOS ONE 17 (5): 1-16.

Objective:

A longitudinal cohort study was carried out to identify potential biomarkers suggestive of bone remodeling and the presence of stomal tissue inflammation. The objective of this study was to investigate trends toward bone and stomal healing.

Whole blood samples were collected from amputee veterans in the PAXgene Blood RNA Tubes and total RNA was isolated, sequenced and analyzed in accordance with manufacturer’s instructions. Sequencing was performed by the High-Throughput Genomics and Bioinformatics Analysis Shared Resource at the Huntsman Cancer Institute at the University of Utah.

Conclusion:

This preliminary study of whole-blood RNA-seq from 10 transfemoral amputees, uncovered two major results:

1) Two inflammatory-related genes (IL33 and IL1RL2) were found to be differentially expressed by 20-fold. These genes may serve as important biomarkers in the serial assessment of stomal wound healing.
2) Two other biomarkers related to bone remodeling (COL2A1 and SOST) were identified. If verified by future studies, these four genes may serve as markers for predicting optimal bone remodeling and stomal tissue healing following the percutaneous osseointegrated implantation procedure.

Craig et al., 2021 “RNA sequencing of whole blood reveals early alterations in immune cells and gene expression in Parkinson’s disease: Nature Aging volume 1, 734–747.

Objectives:

Changes in the blood-based RNA transcriptome have the potential to inform biomarkers of Parkinson’s disease (PD) progression. The authors wanted to gain new insights into the biological pathways in PD for developing diagnostic and prognostic PD biomarkers. Authors sequenced a discovery set of whole-blood RNA species in 4,871 longitudinally collected samples from 1,570 clinically phenotyped individuals from the Parkinson’s Progression Marker Initiative (PPMI) cohort.

Whole Blood was collected in PAXgene Blood RNA Tubes and samples were sent to the biorepository at Indiana University (IU) for RNA isolation and then sent to the HAIB for library preparation and sequencing.

Conclusion:

Participants with PD had significantly altered RNA expression (>2,000 differentially expressed genes). This publicly available transcriptomic dataset, coupled with available detailed clinical data, provided new insights into PD biological processes for developing PD biomarkers.

Adam et al., 2016 “Exome Sequencing Identifies Biallelic MSH3 Germline Mutations as a Recessive Subtype of Colorectal Adenomatous Polyposis”, Am J Hum Genet: 99(2): 337–351.

Objectives:

The authors investigated potential germline mutations in colorectal adenomatous polyposis to uncover hereditary etiology. Exome sequencing was performed in individuals with adenomatous polyposis. Identifying further genetic causes will extend the knowledge of disease mechanisms, biological pathways, and potential therapeutic targets.

Venous blood samples were collected in the PAXgene blood RNA Tubes and total RNA was isolated with the PAXgene Blood RNA Kit in accordance with the manufacturer’s protocol. First-Strand Synthesis System for RT-PCR was carried out and RT-PCR products were separated on agarose gel; individual bands were excised from the gel and eluted DNA was amplified and sequenced.

Conclusion:

Data suggested that MSH3 mutations represents an additional recessive subtype of colorectal adenomatous polyposis. This study identified biallelic pathogenic MSH3 germline mutations as a cause of an inherited tumor syndrome. Thus, MSH3 deficiency might also be of therapeutic relevance for individuals with MSH3-associated polyposis.

Hussain et al 2012., “A Truncating Mutation of CEP135 Causes Primary Microcephaly and Disturbed Centrosomal Function”, Am J Hum Genet: 90(5): 871–878.

Objectives:

Autosomal-recessive primary microcephaly (MCPH) is a rare congenital disorder characterized by intellectual disability, reduced brain and head size. Authors studied a family from northern Pakistan where the two affected children had primary microcephaly at birth using homozygosity mapping.

The PAXgene Blood RNA System was used to extract RNA from patient and control blood samples; Pyrosequencing was done according to manufacturer’s instructions. They sequenced two positional candidate genes and identified a homozygous frameshift mutation in the gene encoding the 135 kDa centrosomal protein (CEP135), located in the linkage interval on chromosome 4, in both affected children. The PAXgene Blood RNA System was used to extract RNA from patient and control blood samples.

Conclusion:

Authors showed that a truncating mutation of CEP135 caused autosomal-recessive primary microcephaly in a Pakistani family.

Zhao et al., 2018 “Evaluation of two main RNA-seq approaches for gene quantification in clinical RNA sequencing: polyA+ selection versus rRNA depletion”, Scientific Reports Volume 8, Article number: 4781

Objective:

Highly abundant riboso mal RNAs (rRNA) are usually removed from total RNA either by positive polyA+ selection or by rRNA depletion (negative selection) before sequencing for efficient transcript/gene detection. Two previous assessments of RNA-seq protocols have commonly used animal samples or cell-line-derived RNAs, but in this study, the two RNA sequencing approaches were investigated in a clinical setting where RNA samples were collected from human blood and colon tissue samples.

Peripheral venous blood samples were collected in PAXgene Blood RNA Tubes. The blood samples were pooled across subjects and replicates were sequenced, using both protocols. Total RNA was isolated from four of the aliquots using a PAXgene Blood RNA Kit according to the manufacturer’s protocol.

Conclusion:

This study evaluated both RNA sequencing approaches and results showed that rRNA depletion captured more unique transcriptome features, whereas polyA+ selection outperformed rRNA depletion with higher exonic coverage and better accuracy of gene quantification.

Harrington et al., 2020 “RNA-Seq of human whole blood: Evaluation of globin RNA depletion on Ribo-Zero library method”, Scientific Reports volume 10, Article number: 6271

Objective:

Highly abundant ribosomal RNAs (rRNA) are usually removed from total RNA either by positive polyA+ selection or by rRNA depletion (negative selection) before sequencing for efficient transcript/gene detection. Two previous assessments of RNA-seq protocols have commonly used animal samples or cell-line-derived RNAs, but in this study, the two RNA sequencing approaches were investigated in a clinical setting where RNA samples were collected from human blood and colon tissue samples.

Conclusion:

Son et al., 2021 “Whole blood RNA sequencing reveals a differential transcriptomic profile associated with cervical insufficiency: a pilot study”, Reproductive Biology and Endocrinology: 19:32

Objective:

This study investigated whether women with Cervical insufficiency (CI) have a differential transcriptomic profile in whole blood to that of controls. CI (formerly called cervical incompetence) is painless cervical dilation resulting in delivery of a live fetus during the second trimester.

Blood samples for qRT-PCR were collected into PAXgene Blood RNA tubes. Total RNA was isolated from the frozen blood samples using a PAXgene Blood RNA Kit according to the manufacturer’s protocol for qRT-PCR validation of differentially expressed gene and RNA was sequenced.

Conclusion:

The authors found that there were significant differences in the whole blood transcriptomic profiles of women with CI compared to those of controls and that different immune responses in women with CI may affect pregnancy outcomes.

Kuiper et al., 2022 “Bridging a diagnostic Kawasaki disease classifier from a microarray platform to a qRT-PCR assay”, Pediatric Research

Objective:

Kawasaki disease (KD) is a systemic vasculitis that mainly affects children under 5 years. The recent discovery of a whole-blood host response classifier that discriminates KD patients from patients with other febrile conditions can aid in early diagnosis of KD. This study bridged this microarray-based classifier to a clinically applicable quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay: the Kawasaki Disease Gene Expression Profiling (KiDs-GEP) classifier. Authors developed a modified one-Step multiplex qRT-PCR assay previously described by Wright et al., 2018.

Whole blood was collected in PAXgene Blood RNA tubes, incubated for 2 h at ambient temperature, frozen at -20 °C within 24 h of collection, and stored at -80°C. Total RNA was isolated using PAXgene Blood RNA Kit according to the manufacturer’s instructions. qRT-PCR and microarray analysis was carried out on all samples.

Conclusion:

Authors were able to develop a more rapid and more cost-efficient qRT-PCR assay, bringing a diagnostic test for KD closer to the hospital clinical laboratory.