Preview for Transcriptomics
1. What is transcriptomics?
First, we should know about the transcriptome. Transcriptome is from the transcription by RNA polymerase II to make messenger RNA and those messenger RNA will be translated into polypeptide to function in the individual cell. The transcriptome is the set of all messenger RNA molecules in one cell or a population of cells. The study of transcriptome is transcriptomics, which is the analysis of sequence of RNA molecules.
2. Relationship between genomics and transcriptomics
The common feature of genomics and transcriptomics is that figure out the specific sequence of DNA or RNA. However, in terms of central dogma of biology field, the DNA has storing the information of protein-coding intact sequence, but it cannot express protein by itself. To express protein, it need to be transcribed first by RNA polymerase. Therefore I guess that the genomics is the basement of transcriptome to understand how biological phenomenon works.
3. What are mRNAs?
There are 3 types of RNA. mRNA is the one of those variation of RNA types, and it is called messenger RNA because when it transcribed, it can transfer its gene information to translation complex to express its protein.
4. How to measure RNA expression?
Measuring RNA expression is available in two parts. One is qPCR(quantitative PCR or real-time PCR) with cDNA synthesis. First, synthesis the complement DNA from mRNA or other RNA sequence by using oligo-dT or random hexamer to bind with the tip of the RNA structure which is play a primer action during the synthesis of cDNA by reverse transcriptase. Reverse tranoscriptase is the enzyme that convert RNA to DNA strand.
After synthesis of cDNA, it should be analyzed by qPCR using cDNA as a template. qPCR is the method that detect the quantity by measuring probe intensity which is combined with primer. When it is reached to certain level, then it caculate back with cycle number.
The other method is that visualizing the localization of mRNA by hybridization method. In situ hybridization is the one of the best method to visualize the expression of mRNA in cell. When we tagged fluorescence probe with RNA oligomer which has complement sequence for target mRNA, then it will be fluorescence in situ hybridization(FISH) method. FISH is a cytogenetic technique that uses fluorescent probes that bind to only those parts of the chromosome with a high degree of sequence complementarity.
5. Relationship between Transcriptome and Proteome.
After transcription of mRNA, then they will be translated by ribosome and tRNA and become newly synthesized polypeptides. Those polypeptides will be modified by other molecules or enzyme to become functional protein. In this stage we can apply the proteomics which is the study of the whole set of proteins. Newly synthesized protein was come from the mRNA, so transcriptome and proteome have very intimate relationship.
6. What is a UTR?
UTR is untranslated region in mRNA. In mRNA structure, there are 5'cap, 5'UTR(5' untranslated region), Coding sequences(Cds), 3'UTR(3' untranslated region), and poly-A-tail. 5'cap and poly-A-tail have function to protect the mRNA sequence. Then, what is the role of 5'UTR or 3'UTR?
Usually, 5'UTR has the recognition of ribosome structure to initiate the translation mechanisms. Recently, in the 5'UTR, there is a conserved structure in most of viruses, which is called, Internal Ribosome Entry Site(IRES) has revealed. IRES is the mimic version of tRNA structure to initiate translation again, after completion of upstream translation.
3'UTR has the role of complementary binding with micro-RNA(miRNA), the one type of non-coding RNA, to regulate the expression of the protein by degradation of mRNA or inhibiting/blocking of translation initiation mechanisms.
7. What is ncRNA ?
ncRNA is non-coding RNA which is different from protein sequence coding RNAs such as, tRNA, rRNA and other kinds of RNA(miRNA, piRNA, etc). tRNA is transferRNA which is transfer amino acids to translating mRNA-ribosomal complex, rRNA is ribosomal RNA to constitue intact ribosome structure. They have a lots of role in translation, splicing, DNA replication, gene regulation, etc. Now, researchers focusing on how these RNAs are affecting to specific biological impact on the organisms.
8. What is poly A ?
Poly-A-tail is the most downstream structure in mRNA. This structure can protect the mRNA from the degradation because mRNA is single stranded, therefore it can be easily degraded.
9. RNA-seq
What to do:
1. Apply Biology learning procedure
Reference
http://www.biomedcentral.com/1471-2164/13/473
https://www.youtube.com/watch?v=UYJfM2-Y7y8 (Youtube by Illumina Gary Schroth)
http://www.saylor.org/content/bio403/assessments/BIO403_Unit_2_2_assessment_RT_PCR_a.html
http://10e.devbio.com/article.php?id=293
https://www.biosearchtech.com/support/education/stellaris-rna-fish
