CHAPTER 1 (RNA EXTRACTION) Flashcards
(19 cards)
what do we need RNA for?
- identifying which genes are active (making RNA)
- finding at what time and in which tissue or condition the genes are active
- measuring how much RNA is produced from a gene (expression levels)
what is RNA
ribonucleic acid
involved in converting the genetic information in DNA into proteins
in retroviruses, RNA is
the genetic material
which part in the RNA that made its structure unstable
the -OH on the 2’ carbon of its ribose.
what can the -OH in RNA do?
attack nearby phosphate bond in its own backbone (intramolecular transesterification) - which breaks the the RNA strand spontaneously esp under heat, alkaline condition or just over time.
RNA nucleotides
pyrimidines:
1. Uracil
2. Cytosine
purines:
1. adenine
2. guanine
location of RNA
nucleus and cytoplasm
location of DNA
nucleus
problems with RNA
- it is chemically unstable
- susceptible to nearly ubiquitous RNA-degrading enzymes or ribonuleases (RNase)
whats RNase
RNAses naturally occurring enzymes that degrade RNA
1. RNases are released upon cell lysis
2. RNases are present on the skin
3. RNases are very difficult to inactivate bcs:
– disulfide bridges conferring stability
– no requirement for divalent cations for
activity
whats the significance of RNase not having requirements for divalent cations for activity?
many enzymes need this to work but RNases dont so they can degrade RNA without needing any extra metal ions.
makes them even harder to stop, removing metal ions wont inactivate them
how to protect sample from endogenous rnases
- Samples should be processed immediately or stored at -70°C until required.
- Inactivation of RNAases by strong denaturing agents like urea, guanidinium hydrochloride, guanidinium isothiocyanate.
how to protect sample from exogenous rnases
- Use DEPC (diethylpyrocarbonate), a strong RNAase inhibitor
- treat water and laboratory utensils with DEPC. DEPC is a suspected carcinogen
- autoclave glassware, solutions and equipments if possible.
- use disposable gloves, disposable plastic materials that must be RNAase free.
what are the methods in RNA extraction
- by triazol (organic extraction)
- spin column
why do we need to isolate mRNA
- mRNA carries the coding info from genes - it tells us which genes are actively being expressed.
- to not let the mRNA drown with other types of RNA
- making cDNA, studying gene expression, or performing transcriptomics (RNA-seq)
how much of mRNA is in total cellular RNA
2.5-5%
what can be used to purify mRNA feom other RNAs?
oligo (dT) probes (short strings of Thymine (T) nucleotides)
whats the function of polyA tail in mRnA molecules
- helps stabilize the mRNA
- to bind with oligo (dT) which is attached to a solid support like beads or column
how to elute mRNA grom oligo (dT) matrix?
by changing the conditions using water or low-salt buffer which weaken A-T and lets mRNA come off.
