LaCava Research Wiki

AIM/HYPOTHESIS

Prior tests of RNase inhibitors raised the question of how reliable and reproducible the Bioanalyzer is for analysis of RNA sample integrity. We will test this by completing biological triplicates and technical duplicates, send to the Genomics facility on different days.

MATERIALS

• Nuclease free extraction buffer (20mM HEPES, pH7.4, 1% TritonX-100, 500mM NaCl, 1x protease inhibitor); RNAsin 1:250 in Extraction, 1:1000 for washing, no RNAsin in buffer for washing beads.
• Scale: 50mg cell powder per IP reaction with 10ul of beads

METHODS

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

After step 3 samples were pooled to get a homogenous sample. Subsequently the sample was devided into 3 smaller samples to get three replicates.

The following samples were delivered twice, on different days to the Genomics facility for analysis:

1. Bio replicate #1
2. Bio replicate #1
3. Bio replicate #2
4. Bio replicate #2
5. Bio replicate #3
6. Bio replicate #3

RESULTS

First analysis

Second analysis

DISCUSSION

As can be seen in the results, there is a lot of varience in the first time the sample was run. Not only do the RIN numbers differ significantly between biological replicates, but even within technical replicates. Furthermore, there was a DNA contamination, caused by the genomics facility.

The second time the samples were analyzed the results look much more trustworthy. They were ran on the bioanalyzer this time compared to TapeStation the first time. Follow up will be to see if we can check RNA integrity by ourselves in the future.