Sample Collection Protocol Effects on Quantification of Gene Expression in Potato Leaf Tissue

New platforms allow quantification of gene expression from large, replicated experiments but current sampling protocols for plant tissue using immediate flash freezing in liquid nitrogen are a barrier to these high-throughput studies. In this study, we compared four sampling methods for RNA extraction for gene expression analysis: (1) the standard sampling method of flash freezing whole leaves in liquid nitrogen immediately upon removal from the plant; (2) incubation of excised leaf disks for 2 min at field temperature followed by flash freezing; (3) incubation of excised leaf disks for 1 h on ice followed by flash freezing; and (4) incubation of excised leaf disks for 1 h at field temperature followed by flash freezing. Gene expression analysis was done for 23 genes using nCounter, and normalization of the data was done using the geometric mean of five housekeeping genes. Quality of RNA was highest for protocol A and lowest for protocol D. Despite some differences in RNA quality, gene expression was not significantly different among protocols A, B, and C for any of the 23 genes. Expression of some genes was significantly different between protocol D and the other protocols. This study demonstrates that when sampling leaf disks for gene expression analysis, the time between tissue removal from the plant and flash freezing in liquid nitrogen can be extended. This increase in time allowable during sampling provides greater flexibility in sampling large replicated field experiments for statistical analysis of gene expression data.     

Evaluation of different storage methods to characterize the fecal bacterial communities of captive western lowland gorillas (Gorilla gorilla gorilla)

Freezing is considered to be the best method for long-term storage of bacterial DNA from feces; however this method cannot be usually applied for samples of wild primates collected in the challenging conditions of the tropical forest. In order to find an alternative conservation method of fecal samples from wild great apes, we compared freezing with other fixation methods. Fecal samples from 11 captive gorillas (Gorilla gorilla gorilla) from three Czech Zoos were stored using freezing, RNA Stabilization Reagent (RNAlater), and 96% ethanol. Subsequently, the samples were examined using culture-independent methods (PCR-DGGE, and Real-time PCR) to qualitatively and quantitatively assess fecal microbiota composition and to compare differences among the storage methods. Noticeably, freezing samples resulted in the highest recoveries of DNA. No significant differences in DNA recovery were found between freezing and using RNAlater; however, significantly lower DNA concentrations were recovered from samples stored in 96% ethanol. Using PCR-DGGE we found that either 96% ethanol, RNAlater or freezing were suitable for preserving bacterial DNA; however fingerprints obtained from RNAlater storage were more similar to those obtained from the frozen method; in comparison to the patterns resulting from storing samples in ethanol. Using qPCR, frozen samples yielded the highest values of bacterial counts, with the exception of Enterobacteriaceae, which showed the highest numbers using samples stored in ethanol. Sequences of amplicons obtained from PCR-DGGE belonged to the families Clostridiaceae, Lactobacillaceae, Staphylococcaceae, and Lachnospiraceae, phylum Firmicutes; however most amplicons showed sequence similarity to previously uncultured microorganisms. Bacteria belonging to the phylum Firmicutes were the most frequently identified species in the fecal bacterial communities of captive western gorillas. The study showed that RNAlater is an optimal storage method when freezing is not possible.     

RNA preservation of Antarctic marine invertebrates

Fifteen species of marine invertebrate commonly occurring in the near-shore environment of Rothera base, Antarctica, were used to test tissue sample storage protocols with regard to preservation of RNA integrity. After animal collection, the tissues were either immediately extracted for RNA or stored at −80°C after having been, either directly flash frozen in liquid nitrogen or preserved in a commercial RNA storage solution, for extraction in the UK. In four cases, direct flash freezing produced enhanced RNA integrity compared with samples in the commercial storage solution. A subset of samples were further tested for the preferred temperature of storage in the commercial reagent. RNA integrity was well preserved at both +4 and −20°C over periods of 2 months, but degradation was rapid in tissues stored at room temperature. Eight out of the fifteen species only produced a single ribosomal band on gel electrophoresis. This survey provides a guide for tissue transport of Polar cold water marine invertebrates.     

Analysis of blood processing conditions to obtain high-quality total RNA from human leukocyte concentrate

Blood samples are used as a biological source to discover biomarkers of hematological and non-hematological disorders. The present study shows the impact of different experimental conditions associated with cell lysis buffer, TRI-reagent protocol and blood cell storage buffer and their correlation with the quantity, quality and Adrenomedullin gene expression levels of total RNA when RT-PCR technique is used. A leukocyte cell bank protocol is also proposed for further mRNA expression analysis using RNAlater as storage buffer. There is evidence that total RNA isolated from leukocyte concentrate stored for 1 month at -70 degrees C did not show significant differences concerning quality, purity and Adrenomedullin gene expression compared with the freshly processed leukocyte sample.     

Allelic imbalance metre (Allim), a new tool for measuring allele‐specific gene expression with RNA‐seq data

Estimating differences in gene expression among alleles is of high interest for many areas in biology and medicine. Here, we present a user‐friendly software tool, Allim, to estimate allele‐specific gene expression. Because mapping bias is a major problem for reliable estimates of allele‐specific gene expression using RNA‐seq, Allim combines two different strategies to account for the mapping biases. In order to reduce the mapping bias, Allim first generates a polymorphism‐aware reference genome that accounts for the sequence variation between the alleles. Then, a sequence‐specific simulation tool estimates the residual mapping bias. Statistical tests for allelic imbalance are provided that can be used with the bias corrected RNA‐seq data.     

Variations in the microbiome due to storage preservatives are not large enough to obscure variations due to factors such as host population,host species,body site,and captivity

The study of the primate microbiome is critical in understanding the role of the microbial community in the host organism. To be able to isolate the main factors responsible for the differences observed in microbiomes within and between individuals, confounding factors due to technical variations need to be removed. To determine whether alterations due to preservatives outweigh differences due to factors such as host population, host species, body site, and habitat, we tested three methods (no preservative, 96% ethanol, and RNAlater) for preserving wild chimpanzee (fecal), wild lemur (fecal), wild vervet monkey (rectal, oral, nasal, otic, vaginal, and penile), and captive vervet monkey (rectal) samples. All samples were stored below ? 20°C (short term) at the end of the field day and then at ? 80°C until DNA extraction. Using 16S rRNA gene sequencing, we show a significant preservative effect on microbiota composition and diversity. Samples stored in ethanol and RNAlater appear to be less different compared with samples not stored in any preservative (none). Our differential analysis revealed significantly higher amounts of Enterococcaceae and Family XI in no preservative samples, Prevotellaceae and Spirochaetaceae in ethanol and RNAlater preserved samples, Oligosphaeraceae in ethanol‐preserved samples, and Defluviitaleaceae in RNAlater preserved samples. While these preservative effects on the microbiome are not large enough to remove or outweigh the differences arising from biological factors (e.g., host species, body site, and habitat differences) they may promote misleading interpretations if they have large enough effect sizes compared to the biological factors (e.g., host population).     

A draft genome sequence of the pulse crop chickpea (Cicer arietinum L.)

Cicer arietinum L. (chickpea) is the third most important food legume crop. We have generated the draft sequence of a desi‐type chickpea genome using next‐generation sequencing platforms, bacterial artificial chromosome end sequences and a genetic map. The 520‐Mb assembly covers 70% of the predicted 740‐Mb genome length, and more than 80% of the gene space. Genome analysis predicts the presence of 27 571 genes and 210 Mb as repeat elements. The gene expression analysis performed using 274 million RNA‐Seq reads identified several tissue‐specific and stress‐responsive genes. Although segmental duplicated blocks are observed, the chickpea genome does not exhibit any indication of recent whole‐genome duplication. Nucleotide diversity analysis provides an assessment of a narrow genetic base within the chickpea cultivars. We have developed a resource for genetic markers by comparing the genome sequences of one wild and three cultivated chickpea genotypes. The draft genome sequence is expected to facilitate genetic enhancement and breeding to develop improved chickpea varieties.     

Genome‐wide characterization and expression analysis of genetic variants in sweet orange

Heterozyosity is an important feature of many plant genomes, and is related to heterosis. Sweet orange, a highly heterozygous species, is thought to have originated from an inter‐species hybrid between pummelo and mandarin. To investigate the heterozygosity of the sweet orange genome and examine how this heterozygosity affects gene expression, we characterized the genome of Valencia orange for single nucleotide variations (SNVs), small insertions and deletions (InDels) and structural variations (SVs), and determined their functional effects on protein‐coding genes and non‐coding sequences. Almost half of the genes containing large‐effect SNVs and InDels were expressed in a tissue‐specific manner. We identified 3542 large SVs (>50 bp), including deletions, insertions and inversions. Most of the 296 genes located in large‐deletion regions showed low expression levels. RNA‐Seq reads and DNA sequencing reads revealed that the alleles of 1062 genes were differentially expressed. In addition, we detected approximately 42 Mb of contigs that were not found in the reference genome of a haploid sweet orange by de novo assembly of unmapped reads, and annotated 134 protein‐coding genes within these contigs. We discuss how this heterozygosity affects the quality of genome assembly. This study advances our understanding of the genome architecture of sweet orange, and provides a global view of gene expression at heterozygous loci.     

Long Term Storage of Dry versus Frozen RNA for Next Generation Molecular Studies

The standard method for the storage and preservation of RNA has been at ultra-low temperatures. However, reliance on liquid nitrogen and freezers for storage of RNA has multiple downsides. Recently new techniques have been developed for storing RNA at room temperature utilizing desiccation and are reported to be an effective alternative for preserving RNA integrity. In this study we compared frozen RNA samples stored for up to one year to those which had been desiccated using RNAstable (Biomatrica, Inc., San Diego, CA) and stored at room temperature. RNA samples were placed in aliquots and stored after desiccation or frozen (at −80°C), and were analyzed for RNA Integrity Number (RIN), and by qPCR, and RNA sequencing. Our study shows that RNAstable is able to preserve desiccated RNA samples at room temperature for up to one year, and that RNA preserved by desiccation is comparable to cryopreserved RNA for downstream analyses including real-time-PCR and RNA sequencing.