LaCava Research Wiki

AIM/HYPOTHESIS

Given the costs of Promega RNasin, we want to test the effectiveness of other, cheaper, RNAse inhibitors. Here, I tested RNasin vs. Ribonucleoside Vanadyl Complex at different concentrations and in both ectopically expressed LINE1 cell line MT302 and tumor sample.

MATERIALS

• Nuclease free extraction buffer (20mM HEPES, pH7.4, 1% TritonX-100, 500mM NaCl, 1x protease inhibitor); RNAse inhibitor at following concentrations.

1. MT302 anti-ORF1 1:250 RNasin EB; 1:1000 RNasin wash (control)
2. MT302 anti-ORF1 Ribonucleoside Vanadyl Complex 10 mM EB+wash; no RNasin
3. MT302 anti-ORF1 Ribonucleoside Vanadyl Complex 10 mM EB+wash; 1:250 RNasin EB; 1:1000 RNAsin wash
4. 162TA anti-ORF1 Ribonucleoside Vanadyl Complex 10 mM EB+wash; no RNasin wash
5. 162TA anti-ORF1 Ribonucleoside Vanadyl Complex 10 mM EB+wash; 1:40 RNasin EB; 1:250 RNasin wash 
6. 162TA anti-ORF1 Ribonucleoside Vanadyl Complex 10 mM EB+wash; 1:250 RNasin EB; 1:1000 RNasin wash 

• Scale: 50mg cell powder per IP reaction with 10ul of beads

METHODS

This experiment was performed according to RNA extraction after cell powder IP.

RESULTS

Two analyses were done on the Bioanalyzer. The first analysis was the first time the Jarvis’ lab instrument was used with our own kit. Given the peculiarity of results, samples were run again, where sample 2 was loaded only once due to limited amount of sample.

Run #1

Run #2

DISCUSSION

The presence of the ribonucleoside vanadyl complex seems to inhibit the detection of the ribosomal subunits, and thus interferes with the readout for RNA integrity. As such, the next step will be to perform an experiment were the vanadyl complex will be depleted right before the RNA extraction by adding EDTA.

Furthermore, the tumor samples are degraded despite the type and amount of RNAse inhibitor used. After discussing with John and Angelica we hypothesized that this might be due to the time in buffer, since direct RNA extraction from powder (without IP) does seem to yield integer RNA. If the vanadyl depletion works, I will test this by doing a time course experiment, varying the time of the sample in the buffer but without an IP. If it turns out that shorter buffer time may yield better RNA, we might shorten the IP time and increase e.g. the amount of beads.