LaCava Research Wiki

AIM/HYPOTHESIS

When we do an IP against ORF1p we pull out full ribosomal complexes. This allows us to use it as a proxy for RNA quality. On advice of Michal Domanski, we decided to spike in EDTA and MgCl2 in our extraction buffer however, to dissociate and stabilize, respecitvely, the ribosomes. In this way we were hoping to see which ribosomal subunit ORF1p preferentially binds to.

MATERIALS

• Extraction buffer
  • 20mM HEPES pH 7.4
  • 500mM NaCl
  • 1% Triton-X100
  • 1X protease inhibitor
  • 1:250 RNasin
  • (25mM EDTA)
  • (10mM MgCl2)
• Wash buffer
  • 20mM HEPES pH 7.4
  • 500mM NaCl
  • 1% Triton-X100
  • 1X protease inhibitor
  • 1:1000 RNasin
  • (25mM EDTA)
  • (10mM MgCl2)

Cell lines:

• N2102Ep
• NTERA2
• MT302 light

Scale: 50mg with 10ul anti-ORF1p beads

Samples

1. N2102Ep -
2. N2102Ep - MgCl2
3. N2102Ep - EDTA
4. NTERA2 -
5. NTERA2 - MgCl2
6. NTERA2 - EDTA
7. MT302 -
8. MT302 - MgCl2
9. MT302 - EDTA

METHODS

IP and RNA extraction according to RNA extraction after cell powder IP.

Then 1ul was loaded on a pico chip and analyzed on the bioanalyzer.

RESULTS

find the bioanalyzer result here

DISCUSSION

No real differnce in both endogenous cell lines, which is to be expected given their low L1 expression. In MT302 however, the large subunit is almost completely gone upon EDTA treatment.