Gene expression profiling

overview of gene expression and transcript profiling

1. Gene expression and Transcript profiling priyanka Mudad M.Sc. Biochemistry central university of Haryana

2. next step after  sequencing a genome determination of the pattern of genes expressed, at the level of transcription, under specific circumstances measurement of the activity (the  expression ) of thousands of genes at once, to create a global picture of cellular function.   Sequence based techniques, like  RNA-Seq , provide information on the sequences of genes in addition to their expression level. expression profile allows one to deduce a cell's type, state, environment, and so forth. Gene expression profiling

3.   DNA  chip or  biochip collection of microscopic DNA spots attached to a solid surface.  measure the  expression  levels of large numbers of genes simultaneously or to  genotype  multiple regions of a genome.  Each DNA spot contains  picomoles  (10 −12   moles ) of a specific DNA sequence, known as  probes  (or  reporters  or  oligos ). These can be a short section of a  gene  or other DNA element that are used to  hybridize  a  cDNA  or cRNA (also called anti-sense RNA) sample (called  target ) under high-stringency conditions.  Probe-target hybridization is usually detected and quantified by detection of  fluorophore -, silver-, or  chemiluminescence -labeled targets to determine relative abundance of nucleic acid sequences in the target. 

4. Hybridization between the cDNA reverse transcribed from a biological sample to a pre-designed complementary DNA probe arranged on a slide, or array, is the basis of DNA microarrays.

5. Major Steps In Microarray Experiment

6. Gene Discovery Disease Diagnosis Drug Discovery Toxicological Research For easing the accessibility to this data, the  National Center for Biotechnology Information  ( NCBI ) has formulated the  Gene Expression Omnibus  or  GEO . It is a data repository facility which includes data on gene expression from varied sources .

7.  cross-hybridization artifacts, poor quantification of lowly and highly expressed genes needing to know the sequence  a priori

8. A/C  whole transcriptome shotgun sequencing

9. It uses  next-generation sequencing  to reveal the presence and quantity of  RNA  in a biological sample at a given moment. RNA-Seq is used to analyze the continuously changing cellular  transcriptome . alternative gene spliced transcripts ,  post-transcriptional modifications ,  gene fusion , mutations / SNPs  and changes in gene expression over time, or differences in gene expression in different groups or treatments. Fragmented cDNA is sequenced, the resulting sequences (“reads”) are mapped and compared to known genomes or transcriptomes.   In addition to mRNA transcripts, RNA-Seq can look at different populations of RNA to include total RNA, small RNA, such as  miRNA ,  tRNA , and  ribosomal profiling . Three most widely used NGS platforms for RNA-seq are SOLiD and Ion Torrent , both marketed by ThermoFisher , and Illumina’s HiSeq .

10. Get your sample Lyse the cells and extract RNA Convert the RNA to cDNA The cDNA pool get sequenced

11. description small\miRNA Degradome- seq Digital gene expression Poly[A]-RNA Seq Total RNA seq Library preparation- RNA selection Size selection- small RNA Poly[A] tail selection- miRNA Poly[A] tail selection- mRNA Poly[A] tail selection- mRNA rRNA depletion - mRNA+IncRNA Sequencing cycles 50 50 50 2 × 100 2 × 100 Single vs. paired end Single end Single end Single end Paired end Paired end Data analysis discovery expression annotation miRNA target ID degradation plots Expression annotation discovery expression annotation discovery expression annotation

12. sequencing

13. Basic steps