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What is used to get rid of RNase? Why do we need to add this?
- - DEPC (Diethylpyrocarbonate) gets rid of RNases
- - We need to get rid of RNases if we want to study RNA! Otherwise, obviously, the RNase is just going to chew up our RNA's that are present! (it is a type of nuclease)
What type of gel do we use for RNA analysis?
RNA analysis uses a Formamide gel
What type of membrane do we use for RNA analysis?
What initiates wound healing?
When damage to a tissue causes its tissue factor to be exposed towards the lumen of the nearby vessel such that it is in contact with its bloodstream. Circulating Factor VII (7) which is always floating around in our system sees this and binds to it, then the whole cascade starts!
How are platelets made?
- Platelets are made by the Megakaryocytes.
- >> The Megakaryocyte just buds up and forms a vesicle that is "exocytosed" out. This vesicle contains cytoplasm that was inside the Megakaryocyte and is packaged along with a bunch of different growth factors that happened to be in that area of the cytoplasm that was packaged up and budded out.
What causes platelet degranulation?
- After Factor 7 gets activated (by binding and releasing from tissue factor) it goes on to convert Prothrombin into Thrombin. Thrombin has a receptor for platelets on it, and upon a platelet binding to this receptor, it degranulates!
- >> After degranulation of the platelet, ALL of the growth factors that were contained within the platelet are released (as the platelet vesicle has now ruptured open)
What do the growth factors released by Platelets (after platelet degranulation) recruit to the wounded site?
- Growth Factors from platelet degranulation recruit:
- - Macrophages
- >> These clean up dead stuff (cells)
- - Leukocytes (specifically, neutrophils)
- >> Neutrophils enter the damaged tissue from the blood stream via neutrophil diapedesis and go on to actively kill live, foreign bacterial cells
- - Mast Cells
What does TGF-ß do?
TGF-ß allows Fibroblasts to proliferate, and it allows them (the fibroblasts) to express scar matrix
What does PDGF (Platelet-derived Growth Factor) do?
PDGF allows for migration within the tissue now (not in the blood) of the fibroblasts to the actual wound site. After the fibroblasts arrive at the wound site, it begins to actually divide (proliferate) in response to TGF-ß that platelets in the area that were degranulated released
What stimulates Maturation of the wound site?
After fibroblasts have started to divide (proliferate), once reaching a critical/threshold amount of fibroblasts in the area, some of the fibroblasts begin to contract (by activating actin-myosin complexes)
When does Endothelial Migration happen?
What is it?
- - Endothelial Migration occurs during the Proliferation stage of wound healing
- - It is the establishment of lateral blood vessels that branch off of the main blood vessel in the damaged area, in response to Angiogenic Growth Factor. This allows for new (small) vessels to provide fresh blood (which contains nutrients, etc.) to the wounded tissue site.
What's the advantage of using In Situ Hybridization over Antibody Binding (Immunocytochemistry)?
In Situ Hybridization lets us bind to small sequences of nucleic acids that would otherwise be too small for an antibody to bind to.
What is the purpose of qRT-PCR?
qRT-PCR is used to (semi-)quantify the amount of mRNA that was present in a sample at the beginning. To fully quantify it we would just need to make a standard curve first (using standards).
What is in Loading Buffer?
- - ß-Mercaptoethanol
- - SDS
- - Glycerol
- - Bromphenol Blue
What is in Running Buffer?
- - Tris-HCl
- - Glycine
- - SDS (ensures that as it runs, the protein remains uniformly negatively charged)
How do you set up a gel sandwich (in Western Blotting/SDS PAGE)?
Sponge : Filter Paper : Gel : Nitrocellulose Membrane : Filter Paper : Sponge
*The Nitrocellulose membrane needs to be placed on the side of the gel that is facing the positively charged anode
What is in Transfer Buffer?
- - Tris-HCl
- - Glycine
- - Methanol
How do you make a cDNA Library?
Add Oligo-dT's (to bind to the Poly-AAA tails) ==> Reverse Transcriptase (to complement the rest of the opposing mRNA) ==> now the cDNA is made
THEN, Taq Polymerase + random Hexamers (small primers) ==> makes a BUNCH of copies of the DNA required to make the original mRNA's protein
How do you make a RNA Clone?
(of one specific mRNA)
- If you already know what you're looking for, you just add the two Primers (one at the very beginning, right after the poly-AAA tail; the other primer at the very end)
- >> we can do this because at this point we already know what the protein is (and thus know its sequence)
How would you see what all mRNA's were being translated at one given time in a cell?
Reverse Transcriptase w/ Oligo-dT primers
What is SYBR Green used for?
- SYBR Green is used to semi-quantify how much double-stranded DNA is present in a cell
- >> it is only semi-quantification because you could have asymmetrical binding of SYBR Green to ds-DNA (ex: 20 on one molecule but only 5 on another one)
When is a Quencher used?
- - We use it when we want to quantify (or semi-quantify) PCR
- >> ex: you can get the ratio of the fluorescence
- >> Type 1: is floating around in the hairpin shape
- >> Type 2: is when it's just straight and the quencher needs to be cleaved off
- - The quencher is attached to the probes (which are at the ends of our primers)
What is Multiplexing and when do you use it?
- - It's basically just our controls (housekeeping genes, etc.) for quantifying
- - You use it with qRT-PCR