Project description:Archival formalin-fixed paraffin-embedded (FFPE) tissue samples hold a wealth of transcriptomic information; however, little is known about potential artifacts. Previously, we identified a consistent shift in global RNA-sequencing profiles between matching frozen and FFPE samples. We hypothesized that this shift was from fixing fresh tissue in formalin. To test this idea, RNA-sequencing was performed on liver samples collected from male mice treated with 600 ppm of a reference chemical (phenobarbital, 600 ppm phenobarbital) or vehicle control for 7 days. Samples were divided into: (1) fresh-frozen (FR); (2) directly fixed in 10% buffered formalin for 18 hours followed by paraffin embedding (FFPE); (3) frozen then fixed as FFPE (FR>FFPE); or (4) frozen then fixed in 70% ethanol followed by paraffin embedding (FR>OH) (n=6/group/condition). Direct fixation resulted in 2946 differentially expressed genes (DEGs), 98% of which were down-regulated. Freezing prior to fixation resulted in ≥95% fewer DEGs vs. FR, indicating that most formalin-derived transcriptional effects occurred with fixation. This was supported by follow-up studies, which identified consistent enrichment in oxidative stress, mitochondrial dysfunction, and transcription elongation pathways with formalin fixation. Notably, formalin fixation in the parent study did not significantly impact chemical response profiles, which were consistent with CAR/PXR activation and 600 ppm phenobarbital exposure. Our results demonstrate distinct transcriptional effects of formalin fixation that could impact gene expression studies using FFPE samples.
Project description:We performed genome-wide 5hmC Methylated DNA Capture (5hMethylCap-seq) on one pooled RCC tissue sample (n=3) and the corresponding matched normal kidney tissue (NAT) (n=3), and we also performed 5hMethylCap-seq on one pooled urine sample obtained from RCC patients (n=52) along with another pooled urine sample obtained from control patients without malignancy (n=65). Global 5hmC levels were dramatically reduced in RCC tissues compared to matched normal adjacent kidney tissues, and although we detected low levels of 5hmC in urine samples, we also observed reduction of 5hmC in urine samples compared to tissue samples. Through assessing histone marked regions we found that 5hmC levels were enriched in H3K9me3 marked repressive genomic regions of normal adjacent kidney compared to RCC tissue tissues. Given the lower 5hmC signal in other genomic regions in cancer tissues, this upregulated 5hmC levels in H3K9me3 marked regions were also clearly identified comparing urine samples from RCC patients to control patients without RCC. We used Caki1 and Caki2 RCC cells to established stable cells with low H3K9me3 expression by knocking down the SUV39H1 gene. We found that low global H3K9me3 causes major upregulation of 5hmC at H3K9me3 marked regions and minor downregulation of 5hmC at genebody regions without change global 5mC and 5hmC levels.
Project description:We performed 10x Genomics single-cell RNA sequencing (scRNA-seq) on 12 urine samples from kidney transplant recipients. These urine samples contain a mixture of donor-derived kidney cells and recipient-derived immune and bladder cells. To our knowledge, this is the first study to apply single-cell transcriptomics to urine samples from kidney allograft recipients.
Project description:miRNAs have been proven to be very useful biomarkers, readily detectable in body fluids, particularly urine may be a valuable source to identify changes in miRNA levels that contribute to better differentiate prostate cancer (PCa) from benign prostate hyperplasia (BPH) cases. In order to characterize microRNA expression in urine samples from PCa, we analyzed expression of 376 microRNAs in 9 samples of PCa and 9 of BPH. The Normalized Ct values were compared between PCa and BPH. Statistical comparisons were made using Mann-Whitney U test, considering two different distributions. We found statiscally differences n expression for 21 miRNAs (Fold change >2 and P value<0.05).
Project description:Gene expression profiling of epidermal samples obtained from sun-exposed and sun-protected body sites from younger (<35 years old) and older (>60 years old) individuals. The Affymetrix U133A plus 2.0 array was used to obtain gene expression data. Samples included 4 younger sun exposed epidermal samples, 4 older sun exposed epidermal samples, 3 younger sun protected epidermal samples, 5 older sun protected epidermal samples.
Project description:miRNAs have been proven to be very useful biomarkers, readily detectable in body fluids, particularly urine may be a valuable source to identify changes in miRNA levels that contribute to better differentiate prostate cancer (PCa) from benign prostate hyperplasia (BPH) cases. In order to characterize microRNA expression in urine samples from PCa, we analyzed expression of 376 microRNAs in 9 samples of PCa and 9 of BPH. The Normalized Ct values were compared between PCa and BPH. Statistical comparisons were made using Mann-Whitney U test, considering two different distributions. We found statiscally differences n expression for 21 miRNAs (Fold change >2 and P value<0.05). For the initial screening of all the studied samples, we selected only those with a concentration above 100 ng/ml, for a total of 9 samples of group of PCa and 9 BPH group. The isolated RNA was evaluated by measuring the absorbance at 260 nm and 280 nm. RNA aliquots from specimens were pooled and reverse transcription (RT) reaction was pweformed. In total, we formed 3 pools from BPH specimens and 3 from PCa ones. The RT product was used to perform a preamplification reaction. The product of preamplification teaction was loaded into the Taqman Homo sapiens microRNA Low density arrays (TLDA) panel A and amplification signal was detected using the 7900 FAST real time thermal cycler (ABI).
Project description:Nonaggressive prostate cancer (NAG-PCa) with Gleason score of 6 is considered as a low-risk disease and does not require clinical interventions. However, current assessment of aggressiveness of PCa is invasive using needle biopsies. Urine is an appealing biospecimen for noninvasive detection of aggressive PCa. Using urine, particularly from easily obtainable from pre-DRE urine specimens, to identify AG-PCa-associated molecular markers is critical for clinical risk stratification. Herein, we acquired quantitative mass spectrometry data for glycoproteins from pre-DRE and post-DRE urine specimens from patients underwent PCa diagnosis with biopsies. Compared with urinary glycoproteins identified from post-DRE urine samples, we confirmed that three previously reported AG-PCa-associated glycoproteins identified in post-DRE urine specimens were also found to be significantly associated with AG disease in pre-DRE urine samples. In addition, new AG-PCa-associated glycoproteins were identified in pre-DRE urine specimens. Our study provides a foundation for further studies of AG-PCa biomarkers using pre-DRE urine specimens.
Project description:Genome-wide gene expression in 66 Rhabdomyosarcoma patients and 16 normal muscle samples were studied using Affymetrix Human Genome U133 Plus 2.0 Array.
Project description:SARS-CoV-2 infection has become a major public health burden and is known to affect many organs with the respiratory system being involved in a majority of cases. Here we undertook a mass spectrometry-based proteomic approach to test whether viral proteins could be detected in urine of patients with COVID-19. Urine samples from 39 patients positive for SARS-CoV-2 by RT-PCR were analysed by mass spectrometry. We detected peptides from the nucleocapsid protein of the SARS-CoV-2 virus in 13 out of 39 urine samples. Thus, our findings suggest that viral proteins present in the urine can be detected by mass spectrometry.