Techniques Flashcards
(7 cards)
Describe cell transfection
Lipofectamine reagant 2000 used.
Lipofectamine reagent contains lipid subunits that can form liposomes in an aqueous environment, which entrap the transfection materials, i.e. DNA plasmids.
The DNA-containing liposomes (with positive charge on their surfaces) can fuse with the negatively charged plasma membrane of living cells, due to the neutral co-lipid mediating fusion of the liposome with the cell membrane, allowing nucleic acid to cross into the cytoplasm and contents to be available to the cell for replication or expression
Positives of RNA transfection
+Endogenous RNA doesn’t generate an immune response
+Transfected cells translate RNA -> protein
Negatives of RNA transfection
- Repeatedly transfecting cells with even a small amount of long RNA can cause cell death unless measures are taken to suppress or evade the innate immune system (but in vitro wouldn’t get immune response anyway?)
- Possibly not representative of cell generated RNA(use diseased human samples in figure 6 to confirm this technique works for them).
Describe linear RNA transfections
Plasmid DNA linearised by PvuII.
Txn. capping and polyaden addition done chemically then cells transfected. Lysates taken 24hrs post transfection (enough time for incorporation)
Describe 5’RACE
Rapid amplification of cDNA Ends.
To obtain the full length sequence of an RNA transcript found within a cell. RACE results in the production of a cDNA copy of the RNA sequence of interest, produced through reverse transcription, followed by PCR amplification of the cDNA copies.
The amplified cDNA copies are then sequenced and, if long enough, should map to a unique genomic region.
Positives of linear RNA transfections
Checks whether there’s a cryptic promotor in the plasmid
Positives of 5’ RACE
RACE can provide the sequence of an RNA transcript from a small known sequence within the transcript to the 5’ end (5’ RACE-PCR)
