Delayed processing of blood samples impairs the accuracy of mRNA-based biomarkers

Background: The transcriptome of peripheral white blood cells (PWBCs) contains valuable physiological information, thus making them a prime biological sample for investigating mRNA-based biomarkers. However, prolonged storage of whole blood samples can alter gene transcript abundance in PWBCs, compromising the results of biomarker discovery. Here, we designed an experiment to interrogate the impacts of delayed processing of whole blood samples on gene transcript abundance in PWBCs. We hypothesized that storing blood samples for 24 hours at 4°C would cause RNA degradation resulting in altered transcriptome profiles. Results: We produced RNA-sequencing data for 30 samples collected from five estrus synchronized heifers (Bos taurus). We quantified transcript abundance for 12,414 protein-coding genes in PWBCs. Analysis of parameters of RNA quality revealed no statistically significant differences (P>0.05) between samples collected from the jugular vein and coccygeal vein, as well as among samples processed after one, three, six, or eight hours. However, samples processed after 24 hours of storage had a lower RNA integrity number value (P=0.03) in comparison to those processed after one hour of storage. Next, we analyzed RNA-sequencing data between samples using those processed after one hour of storage as the baseline for comparison. Interestingly, evaluation of 3/5 prime bias revealed no differences between genes with lower transcript abundance in samples stored for 24 hours relative to one hour. In addition, sequencing coverage of transcripts was similar between samples from the 24-hour and one-hour groups. We identified four and 515 genes with differential transcript abundance in samples processed after storage for eight and 24 hours, respectively, relative to samples processed after one hour. Conclusions: The PWBCs respond to prolonged cold storage by increasing genes related to active chromatin compaction which in turn reduces gene transcription. This alteration in transcriptome profiles can impair the accuracy of mRNA-based biomarkers. Therefore, blood samples collected for mRNA-based biomarker discovery should be refrigerated immediately and processed within six hours post sampling.

Evaluation of reference genes and characterization of the MYBs in xylem radial change of Chinese fir stem

The radial change (RC) of tree stem is the process of heartwood formation involved in complex molecular mechanism. Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.), an evergreen species, is an important fast-growing timber tree in southern China. In this study, the top four stable genes (IDH, UBC2, RCA and H2B) were selected in RC tissues of 15 years old Chinese fir stem (RC15) and the genes (H2B, 18S, TIP41 and GAPDH) were selected in RC tissues of 30 years old Chinese fir stem (RC30). The stability of the reference genes is higher in RC30 than in RC15. Sixty-one MYB transcripts were obtained on the PacBio Sequel platform from woody tissues of one 30 years old Chinese fir stem. Based on the number of MYB DNA-binding domain and phylogenetic relationships, the ClMYB transcripts contained 21 transcripts of MYB-related proteins (1R-MYB), 39 transcripts of R2R3-MYB proteins (2R-MYB), one transcript of R1R2R3-MYB protein (3R-MYB) belonged to 18 function-annotated clades and two function-unknown clades. In RC woody tissues of 30 years old Chinese fir stem, ClMYB22 was the transcript with the greatest fold change detected by both RNA-seq and qRT-PCR. Reference genes selected in this study will be helpful for further verification of transcript abundance patterns during the heartwood formation of Chinese fir.

Functional Identification of ICE Transcription Factors in Rubber Tree

ICE (inducer of CBF expression) is a positive regulator of cold signaling pathway in plants. Identification of ICE transcription factors is important for the sustainable development of the natural rubber planting industry in nontraditional regions where sudden cold waves often occur. In this study, five ICE genes were isolated from genome of rubber tree (Hevea brasiliensis Muell. Arg.) for analysing tolerance to cold stress. They shared an ICE-specific region in the highly conserved bHLH-ZIP domain and were localized in the nucleus. The HbICEs were different in transcript abundance and expression patterns in response to cold and drought stresses and among different rubber tree clones. Generally, the expression level of HbICEs was significantly higher in the cold-tolerant rubber tree clones than that in the cold-sensitive rubber tree clones. Overexpression of HbICE1, HbICE2, and HbICE4 significantly enhanced the cold tolerance of transgenic Arabidopsis and tobacco, which showed a significant increase in chlorophyll content and decrease in relative water content and conductivity at the early stage of cold stress in comparison with wild-type plants. Furthermore, overexpression of HbICE2 and HbICE4, but also HbICE1 enhanced drought tolerance in transgenic Arabidopsis. The cold tolerance of rubber tree clones is positively controlled by the expression level of HbICE1, HbICE2, and HbICE4.

Transcriptomic Analysis of Sex-Associated DEGs in Female and Male Flowers of Kiwifruit (Actinidia deliciosa [A. Chev] C. F. Liang & A. R. Ferguson)

Kiwifruit (Actinidia deliciosa [A. Chev.], C.V. Liang & A. R. Ferguson, 1984) is a perennial plant, with morphologically hermaphroditic and functionally dioecious flowers. Fruits of this species are berries of great commercial and nutritional importance. Nevertheless, few studies have analyzed the molecular mechanisms involved in sexual differentiation in this species. To determine these mechanisms, we performed RNA-seq in floral tissue at stage 60 on the BBCH scale in cultivar ‘Hayward’ (H, female) and a seedling from ‘Green Light’ × ‘Tomuri’ (G × T, male). From these analyses, we obtained expression profiles of 24,888 (H) and 27,027 (G × T) genes, of which 6413 showed differential transcript abundance. Genetic ontology (GO) and KEGG analysis revealed activation of pathways associated with the translation of hormonal signals, plant-pathogen interaction, metabolism of hormones, sugars, and nucleotides. The analysis of the protein-protein interaction network showed that the genes ERL1, AG, AGL8, LFY, WUS, AP2, WRKY, and CO, are crucial elements in the regulation of the hormonal response for the formation and development of anatomical reproductive structures and gametophytes. On the other hand, genes encoding four Putative S-adenosyl-L-methionine-dependent methyltransferases (Achn201401, Achn281971, Achn047771 and Achn231981) were identified, which were up-regulated mainly in the male flowers. Moreover, the expression profiles of 15 selected genes through RT-qPCR were consistent with the results of RNA-seq. Finally, this work provides gene expression-based interactions between transcription factors and effector genes from hormonal signaling pathways, development of floral organs, biological and metabolic processes or even epigenetic mechanisms which could be involved in the kiwi sex-determination. Thus, in order to decode the nature of these interactions, it could be helpful to propose new models of flower development and sex determination in the Actinidia genus.

Genome-Wide Identification and Analysis of Cell Cycle Genes in Betula pendula

Research Highlights: This study identified the cell cycle genes in birch that likely play important roles during plant growth and development. This analysis provides a basis for understanding the regulatory mechanism of various cell cycles in Betula pendula. Background and Objectives: The cell cycle factors not only influence cell cycle progression together, but also regulate accretion, division and differentiation of cells, and then regulate growth and development of plant. In this study, we identified the putative cell cycle genes in B. pendula genome, based on the annotated cell cycle genes in A. thaliana. It could serve as a foundation for further functional studies. Materials and Methods: The transcript abundance was determined for all the cell cycle genes in xylem, root, leaf and flower tissues using RNA-seq technology. Results: We identified 59 cell cycle gene models in the genome of B. pendula, 17 highly expression genes among them. These genes were BpCDKA.1, BpCDKB1.1, BpCDKB2.1, BpCKS1.2, BpCYCB1.1, BpCYCB1.2, BpCYCB2.1, BpCYCD3.1, BpCYCD3.5, BpDEL1, BpDpa2, BpE2Fa, BpE2Fb, BpKRP1, BpKRP2, BpRb1 and BpWEE1. Conclusions: We identified 17 core cell cycle genes in the genome of birch by combining phylogenetic analysis and tissue specific expression data.

Direct RNA sequencing of Respiratory Syncytial Virus infected human cells generates a detailed overview of RSV polycistronic mRNA and transcript abundance

To characterize species of viral mRNA transcripts generated during respiratory syncytial virus (RSV) infection, human fibroblast-like MRC5 lung cells were infected with subgroup A RSV for 6, 16 and 24 hours. Total RNA was harvested and polyadenylated mRNA was enriched and sequenced by direct RNA sequencing on an Oxford nanopore device. This yielded over 150,000 direct mRNA transcript reads which were mapped to the viral genome and analysed to determine relative mRNA levels of viral genes using our in-house ORF-centric pipeline. We were also able to examine frequencies with which polycistronic readthrough mRNAs were generated and to assess the length of the polyadenylated tails for each group of transcripts. We show that there is a general but non-linear decline in gene transcript abundance across the viral genome, as predicted by the model of RSV gene transcription. However, the decline in transcript abundance is not consistent. We show that the polyadenylate tails generated by the viral polymerase are similar in length to those generated by the host cells polyadenylation machinery and broadly declined in length for most transcripts as infection progressed. Finally, we observed that the steady state abundance of transcripts with very short polyadenylate tails is much less for N, SH and G transcripts compared to NS1, NS2, P, M, F and M2 which may reflect differences in mRNA stability and/or translation rates.

Time-resolved assessment of single-cell protein secretion by sequencing

Secreted proteins play critical roles in cellular communication and functional orchestration. Methods enabling concurrent measurement of cellular protein secretion, phenotypes and transcriptomes are still unavailable. Here, we describe time-resolved assessment of protein secretion from single cells by sequencing (TRAPS-seq). Released proteins are trapped onto cell surface via affinity matrices, and the captured analytes together with phenotypic markers can be probed by oligonucleotide-barcoded antibodies and simultaneously sequenced with transcriptomes. We used TRAPS-seq to interrogate secretion dynamics of pleiotropic cytokines (IFN-γ, IL-2 and TNF-α) of early activated human T lymphocytes, unraveling limited correlation between cytokine secretion and its transcript abundance with regard to timing and strength. We found that early central memory T cells with CD45RA expression (TCMRA) are the most effective responders in multiple cytokine secretion, and polyfunctionality involves unique yet dynamic combinations of gene signatures over time. TRAPS-seq presents a useful tool for cellular indexing of secretions, phenotypes, and transcriptomes at single-cell resolution.

Dual transcriptomics to determine interferon-gamma independent host response to intestinal Cryptosporidium parvum infection

Animals with a chronic infection of the parasite Toxoplasma gondii are protected against lethal secondary infection with other pathogens. Our group previously determined that soluble T. gondii antigens (STAg) can mimic this protection and be used as a treatment against several lethal pathogens. Because treatments are limited for the parasite Cryptosporidium parvum , we tested STAg as a C. parvum therapeutic. We determined that STAg treatment reduced C. parvum Iowa II oocyst shedding in IFNγ-KO mice. Murine intestinal sections were then sequenced to define the IFNγ independent transcriptomic response to C. parvum infection. Gene Ontology and transcript abundance comparisons showed host immune response and metabolism changes. Transcripts for type I interferon responsive genes were more abundant in C. parvum infected mice treated with STAg. Comparisons between PBS or STAg treatments showed no significant differences in C. parvum gene expression. C. parvum transcript abundance was highest in the ileum and mucin-like glycoproteins and the GDP-fucose transporter were among the most abundant. These results will assist the field in determining both host- and parasite-directed future therapeutic targets.

Genome-Wide Identification and Analysis of Cell Cycle Genes in Betula pendula

Research Highlights: This study identified the cell cycle genes in birch that likely play important roles during plant growth and development. This provides a basis for understanding the regulatory mechanism of various cell cycles in Betula pendula. Background and Objectives: The cell cycle factors not only influence cell cycle progression together, but also regulate accretion, division and differentiation of cells, and then regulate growth and development of plant. In this study, we identified the putative cell cycle genes in B. pendula genome, based on the annotated cell cycle genes in A. thaliana. It could serve as a foundation for further functional studies. Materials and Methods: The transcript abundance was determined for all the cell cycle genes in xylem, root, leaf and flower tissues using RNA-seq technology. Results: We identified 59cell cycle gene models in the genome of B. pendula, 17 highly expression genes among them. These genes were BpCDKA.1, BpCDKB1.1, BpCDKB2.1, BpCKS1.2, BpCYCB1.1, BpCYCB1.2, BpCYCB2.1, BpCYCD3.1, BpCYCD3.5, BpDEL1, BpDpa2, BpE2Fa, BpE2Fb, BpKRP1, BpKRP2, BpRb1 and BpWEE1. Conclusions: We identified 17 core cell cycle genes in the genome of birch by combining phylogenetic analysis and tissue specific expression data.