Correlation of Cry1Ac mRNA and protein abundance in transgenic Gossypium hirsutum plant.

Transcription and translation in eukaryotes are distinct processes of the molecular cascade leading to protein production from genetic material. However, establishing correlation between mRNA expression and protein abundance, the end results of the two processes of central dogma, remains a challenge. For transgenic plants, such correlation between mRNA and protein expression serves as a guide to design the transgene, in particular the choices of promoter and codon usage to ensure stable expression of the target protein in relevant tissues under various stress conditions. To elucidate level of mRNA-protein correlation in a commercial transgenic cotton plant Gossypium hirsutum, Bollgard II® (MON15985), we present the results of Cry1Ac protein expression correlating with corresponding mRNA levels. Protein was quantitated using a home-grown validated ELISA assay with a monoclonal-polyclonal antibody pair, whereas mRNA level was detected by a real-time quantitative PCR assay using standardized reference genes. Our results indicate that protein and mRNA levels are highly correlated in the leaves, but not in squares and stem. The correlations seem to be consistent between young and mature leaves and increase over time of harvesting of samples from months 1-3. These findings demonstrate that transcript level measurement could serve as a proxy to protein abundance for this commercially important cotton species, particularly for leaf tissues which are the most vulnerable organs to cotton bollworms and other pathogens. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02828-2.

Relative quantification of BCL2 mRNA for diagnostic usage needs stable uncontrolled genes as reference.

Dysregulation of BCL2 is a pathophysiology observed in haematological malignancies. For implementation of available treatment-options it is preferred to know the relative quantification of BCL2 mRNA with appropriate reference genes. For the choice of reference genes-(i) Reference Genes were selected by assessing variation of >60,000 genes from 4 RNA-seq datasets of haematological malignancies followed by filtering based on their GO biological process annotations and proximity of their chromosomal locations to known disease translocations. Selected genes were experimentally validated across various haematological malignancy samples followed by stability comparison using geNorm, NormFinder, BestKeeper and RefFinder. (ii) 43 commonly used Reference Genes were obtained from literature through extensive systematic review. Levels of BCL2 mRNA was assessed by qPCR normalized either by novel reference genes from this study or GAPDH, the most cited reference gene in literature and compared. The analysis showed PTCD2, PPP1R3B and FBXW9 to be the most unregulated genes across lymph-nodes, bone marrow and PBMC samples unlike the Reference Genes used in literature. BCL2 mRNA level shows a consistent higher expression in haematological malignancy patients when normalized by these novel Reference Genes as opposed to GAPDH, the most cited Reference Gene. These reference genes should also be applicable in qPCR platforms using Taqman probes and other model systems including cell lines and rodent models. Absence of sample from healthy-normal individual in diagnostic cases call for careful selection of Reference Genes for relative quantification of a biomarker by qPCR.BCL2 can be used as molecular diagnostics only if normalized with a set of reference genes with stable yet low levels of expression across different types of haematological malignancies.

Usage of Graphene Oxide in Fluorescence Quenching-Linked Immunosorbent Assay for the Detection of Cry2Ab Protein Present in Transgenic Plants.

Graphene oxide-based sensor technologies in various detection platforms have been adopted in multiple dimensions. Most of the applications in combination with other materials such as gold, silver, enzymes, and so forth are read as electrical, electrochemical, impedance, and fluorescence signals. We report the development of a novel and simple fluorescence quenching-based immunoassay platform that provides quantitative binding sites for the Cry2Ab protein content present in the transgenic cotton (Gossypium hirsutum) plant. In this assay, the graphene oxide-conjugated anti-Cry2Ab antibody serves as the binding site for the analyte Cry2Ab protein, which forms a complex with a second anti-Cry2Ab fluorescein isothiocyanate (FITC)-conjugated antibody. This complex acts as the reaction center of this platform where the graphene oxide quenches the fluorescence signal of the FITC molecule. This microtiter plate-based method currently works at a sensitivity of 0.78 ng /ml, which can further be improved.

Genome wide search to identify reference genes candidates for gene expression analysis in Gossypium hirsutum.

BACKGROUND: Cotton is one of the most important commercial crops as the source of natural fiber, oil and fodder. To protect it from harmful pest populations number of newer transgenic lines have been developed. For quick expression checks in successful agriculture qPCR (quantitative polymerase chain reaction) have become extremely popular. The selection of appropriate reference genes plays a critical role in the outcome of such experiments as the method quantifies expression of the target gene in comparison with the reference. Traditionally most commonly used reference genes are the "house-keeping genes", involved in basic cellular processes. However, expression levels of such genes often vary in response to experimental conditions, forcing the researchers to validate the reference genes for every experimental platform. This study presents a data science driven unbiased genome-wide search for the selection of reference genes by assessing variation of > 50,000 genes in a publicly available RNA-seq dataset of cotton species Gossypium hirsutum. RESULT: Five genes (TMN5, TBL6, UTR5B, AT1g65240 and CYP76B6) identified by data-science driven analysis, along with two commonly used reference genes found in literature (PP2A1 and UBQ14) were taken through qPCR in a set of 33 experimental samples consisting of different tissues (leaves, square, stem and root), different stages of leaf (young and mature) and square development (small, medium and large) in both transgenic and non-transgenic plants. Expression stability of the genes was evaluated using four algorithms - geNorm, BestKeeper, NormFinder and RefFinder. CONCLUSION: Based on the results we recommend the usage of TMN5 and TBL6 as the optimal candidate reference genes in qPCR experiments with normal and transgenic cotton plant tissues. AT1g65240 and PP2A1 can also be used if expression study includes squares. This study, for the first time successfully displays a data science driven genome-wide search method followed by experimental validation as a method of choice for selection of stable reference genes over the selection based on function alone.

DenHunt - A Comprehensive Database of the Intricate Network of Dengue-Human Interactions.

Dengue virus (DENV) is a human pathogen and its etiology has been widely established. There are many interactions between DENV and human proteins that have been reported in literature. However, no publicly accessible resource for efficiently retrieving the information is yet available. In this study, we mined all publicly available dengue-human interactions that have been reported in the literature into a database called DenHunt. We retrieved 682 direct interactions of human proteins with dengue viral components, 382 indirect interactions and 4120 differentially expressed human genes in dengue infected cell lines and patients. We have illustrated the importance of DenHunt by mapping the dengue-human interactions on to the host interactome and observed that the virus targets multiple host functional complexes of important cellular processes such as metabolism, immune system and signaling pathways suggesting a potential role of these interactions in viral pathogenesis. We also observed that 7 percent of the dengue virus interacting human proteins are also associated with other infectious and non-infectious diseases. Finally, the understanding that comes from such analyses could be used to design better strategies to counteract the diseases caused by dengue virus. The whole dataset has been catalogued in a searchable database, called DenHunt (http://proline.biochem.iisc.ernet.in/DenHunt/).