Question

Design an experiment using RNA sequencing to determine the changes in the transcriptome (global gene expression) of human cells in response to infection with the influenza virus. For full credit, your experiment must be explained in detail and have an appropriate control.

Answer 1

ANSWER )

Global gene expression analysis provides quantitative information about the population of RNA species in cells and tissues.Global gene expression analysis uses DNA microarrays, RNA-Seq, and other methods to measure the levels of RNA species in biological systems. DNA microarrays, which have been most frequently used for expression analysis, consist of millions of individual oligonucleotide probes fixed to a solid surface. The oligonucleotide probes typically have sequences representative of known RNA species and are generally used to quantitate the relative levels of RNA species that hybridize to the probes. Massively parallel sequencing technologies, developed more recently, provide a measure of the frequency of RNA species through sequencing of RNA-derived cDNA populations. Other approaches, such as digital molecular barcoding, represent a fusion of the hybridization and counting approaches.

IAV (INFLUENZA VIRUS ) mainly targets the epithelial cells of the respiratory tract, but can also infect macrophages and dendritic cells. The innate immune system initiates host defenses against IAV infection, which is triggered by pattern recognition receptors (PRRs) that recognize viruses or their genetic material. This recognition results in appropriate antiviral responses, including the production of a variety of cytokines and the induction of inflammatory and adaptive immune responses

EXPERIMENT

• INFECTION OF HINI1 VIRUS (influneza virus )TO THE SPECIFIC HUMAN CELL THAT IS 293T (293T (or HEK 293T is a human cell line, derived from the HEK 293 cell line, that expresses a mutant version of the SV40 large T antigen.) AND CONTROL CELL LINE WHICH WAS NOT INFECTED BY H1N1
• EXTRACTION OF RNA : The cells can be washed in  phosphate-buffered saline (PBS) and incubated in DMEM with or without FBS at 37°C in 5% CO2. The infected cells will be  collected and used for RNA extraction. Total RNA extracted can be done by using the RNeasy Mini kit (Qiagen). RNA quality was assessed by analysis of the rRNA band integrity.
• Total RNA will be  treated with RNase-free DNase I. Further cDNA LIBRARY WILL BE PREPARED
• RNA SEQUENCING CAN BE DONE THROUGH llumina HiSeq 4000 platform to generate 150-bp paired-end reads.
• Ingenuity pathway analysis software (Ingenuity System) Wwill be  used for DIFFERENTIAL EXPRESSION OF GENE (DEG analysis) and the false discovery rate (FDR)-adjusted p < 0.05 will be considered to indicate DEGs. Genes with ≥ 2-fold changes would be selected for further analysis. Gene ontology (GO) functional classifications Can be  determined using Blast2GO software, and the enriched gene functional categories will further classified by GO analysis with p < 0.05.
• Further To investigate possible biological interactions of DEGs and determine important functional networks, DEG datasets will be  imported into the Ingenuity pathway analysis (IPA) tool. To identify functional groups and components of processes that are common and unique between the responses of HEK293T infected and non infected cell line, the statistical enrichment of canonical pathways identified by IPA (p < 0.01) can be calculated.
• To validate the differential expression profiles of genes obtained by RNA-seq analysis, quantitative RT-PCR Can be performed on selected DEGs

HOPE YOU UNDERSTAND