Viral RNA Extraction from Infected Tissues
Instrument: Precellys® 24 Dual
Sample: Mouse murine pancreas (organs in which the virus replicates)
Protocol:
- Precellys kits: CK28 (big ceramic beads)
- Sample : 30-75mg of murine pancreas
- Buffer : volume is adjusted in order to get a 5% w/v homogenate
- Run the Precellys®24 at 6500 rpm, 3x5 sec.
Results:
After homogenizing the murine pancreas, Leuven University detects :
- Firstly the viral RNA (1) The homogenization in lysis buffer RLT is performed, and worked further with the supernatant to extract the viral RNA. Graph (1) presents the evolution of viral RNA in the pancreas of viral-induced mice. It is expressed as “copies viral RNA per copy beta actin” (RT-QPCR after total RNA extraction).
Secondly replicating the infectious virus(2) In the second try (2) the homogenization in growth medium, (MEM, Gibco), is performed using saline buffer. The supernatant is then used to titrate on a cell culture (96-well plates, and the titer of the virus is determined (expressed as CCID50).
Graph(2) presents the evolution of infectious virus: the titer of infectious virus is expressed as “-log CCID50/ml” where CCID50 stands for “Cell Culture Infective Dose 50%”, which is a measure to express the titer of a virus stock.
