METHODS OF TRANSCRIPTOME ANALYSIS....pptx

METHODS OF TRANSCRIPTOME ANALYSIS.pptx

1. METHODS OF TRANSCRIPTOME ANALYSIS SUBMITTED TO RISHIKA P.S DEPARTMENT OF BOTANY ST.TERESAS COLLEGE SUBMITTED By MANEESHA NATH N.M ROLL NO: 11 2 ND M.Sc. BOTANY 1

2. The transcriptome, is the complete set of all RNA molecules (mRNA, rRNA, RNA intron etc) in a cell, a population of cells or in an organism at a given time. Changes with time. After the genome has been sequenced, transcriptome analysis allows us to understand the expression of genome at the transcription level, which provides information on gene structure, regulation of gene expression, gene product function, and genome dynamics. Study of transcriptome is known as transcriptomics. TRANSCRIPTOME 2

3. The genome content and genes remain almost same in all the cells of an organism but for responding to specific environmental change or developmental stage different tissues or the cells needs to decode this genomic information suitably . So this specific set of genes are transcribed to rnawhich are short livedmessenger molecules. As transcriptome referred to expressed part of genome, it also known as functional genome. While transcriptomics is most commonly applied to the mRNAs, the coding transcripts, transcriptomics also provides important data regarding content of the cell noncoding RNAs, including rRNA, tRNA, lncRNA, siRNA, and others. 3 3

4. Transcriptomics is the study of RNA in any of its forms. A study of transcriptome elucidates the complex interactions which generally take place among the transcripts before these are translated. Also an independent analysis of transcriptome in thousand of cell types organs and tissues elucidates regulation of gene expression in time and space through the study of relative abundance of different individual transcripts . TRANSCRIPTOMICS 4

5. TRANSCRIPTOMIC AIMS To catalogue all species of transcripts, including mRNAs, non coding RNAs and small RNAs To determine the transcriptional structure of genes in terms of their start sites, 5'and 3' ends, and other post transcriptional modifications To quantify the changing expression levels of each transcript during development and under different conditions 5

6. TRANSCRIPTOME ANALYSIS There are four methods to analyse a transcriptome Hybridization based Microarray Northern Blotting SAGE Sequencing based RNA-seq 4 6

7. Hybridization based Microarray Microarray is a tool which can detect the expression of thousands of genes simultaneously They also referred as gene chips Slide has many spots containing specific known DNA oligomers which represents entire genome of an organism and this act as a probe to detect gene expression 7

8. HYBRIDIZATION BASED In microarray a predetermined set of probes representing sequence of gene fragments are fixed on a solid chip . The major limitation of this technique is that gene sequence needs to be known for designing the chip . thus for non model species that like genomic sequence information microarray is not viable option as the chips are not readily available and needs to be designed and designing a chip is very costly. 8

9. The technology has been developed in several variants but in the following we only discuss the two most popular: "two colour " (or cDNA or two-channel) microarrays "one colour " (or oligonucleotides or one-channel) microarrays. Two colour microarrays are based on the competitive hybridization of two samples each of which has been labeled with a different fluorescent dye (e.g. red or green). After hybridization, the array is exposed to red and green laser light the array emits fluorescence proportional to the quantity of RNA the image produced is scanned yielding after some corrections a value which represents the expression of one sample relative to the other. 9

10. One channel microarrays are based on RNA of one sample which has been labeled with a fluorescent dye and hybridized to a single array where millions of copies of short (around 24 base pairs) oligonucleotide probes representing all known genes (several probes for gene form a “ probeset ” ) have been synthesized. After exposition to laser light and scanner the intensity of each location is measured yielding a value which represents an absolute measure of expression . 10

11. Slide scanning: The image of hybridized array is captured using a laser scanner. Two wavelengths of laser beams are used to excite the red and green flourescent dyes. A photomultiplier tube detects the flourecence. The two florescent images from the scanner are then overlaid to create a composite image which indicate the relative expression of each gene. The colour intensity measure of the gene expression levels. 11

12. DATA ANALYSIS :- The array signals are then converted to numbers and are reported as ratios between the two colors. This ratio is a measure of the gene expression changes between two samples. Micro scanners are normally provided with software programs to carry out microarray image analysis. There are also a number of free processing software programs available on the internet. Eg: ScanAlyze , ArrayDB etc. 12

13. SAGE- serial analysis of gene expression Serial analysis of gene expression is a highly efficient technology that can give a global expression of profile of a particular type of cell or tissue. PRINCIPLES: 1) A short sequence tags in a defined position in cDNA that contain sufficient information to uniquely identify a transcript 2) the concatenation of tag which allows for efficient sequence based analysis of transcription 3) The tags are then concatenated by ligation with other tags, amplified in a bacterial host and then sequenced 4) The number of times that a specific sequence tag is found determines the relative abundance of the transcript in that sample. 13

14. STEPS INVOLVED 1) isolate mRNA and perform reverse transcription biotinylation of cDNA 14

15. 2) mix cDNA with streptavidin bead(these are superparamagnetic particles covalently coupled to ahighly pure form of streptavidin) this beads will bind to the biotin cDNA complex 15

16. cDNA is cleaved using restriction endonuclease called an anchoring enzyme. Result of this cleavage is that the beads are bound to cDNA fragments of the various lengths with the same sequence at their exposed end. 16

17. Cleaved cDNA that is no longer bound to the bead is now removed by rinsing Remaining bound cDNA is divided into two solutions Add A & B adaptors (either A or B to each) Sticky ends containg anchoring enzyme cut site Restriction enzyme site- tagging enzyme ( downstream) RE used to cleave the cDNA nearly 15bp downstream These remove cDNA from the beads to create a short gun of around 11 nucleotides 17

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20. sticky ends of tags are repaired using DNA polymers This cDNA tag fragments are ligated to form ditags 20

21. Adaptors are removed using Anchoring enzymes Amplification in bacteria 21

22. The amplified sequences are isolated and sequenced using modern high throughput DNA sequencers Sequences can be analysed with computer programs which quantify the recurrence of individual tags 22

23. Northern blotting Northern blot is a technique based on the principle of blotting for the analysis of specific RNA in a complex mixture. The quantity of mRNA transcript for a single gene directly reflects how much transcription of that gene has occurred Tracking of that quantity will therefore indicate how vigorously a gene transcribed or expressed 23

24. Used to visualise differences in quantity of mRNA produced by different groups of cells at different time Different fragments of mRNA are separated from one another via gel electrophoresis and transferred to a filter or other solid support using a technique known as blotting To identify the desired mRNA single standard complimentary RNA that is labelled with the radioactive molecule is allowed to hybridize. Later when the filter paper is placed against X ray film radioactivity in the probe will expose the film thereby making marks on it The intensity of the resulting marks, called bands will tell how much mRNA was in the sample which is direct indicator of how strongly the gene of interest is expressed 24

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26. RNA Sequencing (RNA-Seq) RNA Sequencing (RNA-Seq) is a revolutionary technique for transcriptome-wide analysis of gene expression. Given its high accuracy and sensitivity for measuring expression, it has become the standard for studying transcriptomic dynamics and identifying differences in expression between tissues, physiological stages, diseases and a wide range of other experimental designs. 26

27. This approach offers a number of advantages Provides sensitive, accurate measurement of gene expression does not require predesigned probes Generates both qualitative and quantitative data Reveals the full transcriptome, not just a few selected transcripts Can be applied to any species, even if a reference sequence is not available 27

28. Double stranded DNA is more stable than RNA and can be easily amplified and modified. 28

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30. Sequence the library The machine has flourescent probe that are colour coded according to the type of nucleotide they can bind 30

31. The probes are attached to the first base in each sequence. 31

32. First line is the unique id for the sequence Bases called for the sequenced fragment ‘+’ Sign Quality score for each base in the fragment 47 32

33. REFERENCES Sanchez-Pla, A., Reverter , F., Ruíz de Villa, M. C., & Comabella , M. (2012). Transcriptomics: mRNA and alternative splicing. Journal of Neuroimmunology . Wang, Z., Gerstein, M., & Snyder, M. (2009). RNA-Seq: a revolutionary tool for transcriptomics. Nature Reviews Genetics, 10(1), 57-63 https://www.frontiersin.org/articles/10.3389/fgene.2018.00636/full https://youtu .be/Ou3ga39SVfQ 33

34. THANK YOU 34