Tested RNA extractions using Ambion RiboPure Kit Protocol RNA Extraction Protocol RiboPure Kit

Sample list used for comparisons with Directzol Kit

• EPI-309 S-309 = siphon tissue only stored in RNAlater, ground to powder on liquid nitrogen on 20160912, and stored at -80
• EPI-309 M-309 = whole body and some siphon tissue stored in RNAlater, ground to powder on liquid nitrogen on 20160912, and stored at -80
• Geoduck10 G-10 (tissue from Grace) = adult tissue stored at -80 and extracted immediately after removal from freezer
• EPI-56 = whole body stored at -80, ground to powder on liquid nitrogen on 20160912, and stored at -80
• EPI-103 = whole body tissue stored in RNAlater, ground to powder on liquid nitrogen on 20160912, and stored at -80
• EPI-303
• EPI-302
• EPI-299
• EPI-298
• EPI-290
• EPI-289

Extraction Procedure

• Used ~10mg plastic scoops to transfer 2-4 scoops (~40mg) of ground tissue to 0.5ml TRI Reagent
• Homogenized again with teflon pestles in microfuge tubes with TRI Reagent
• Proceeded with RNA Extraction Protocol RiboPure Kit
• After initial centrifugation, yielded ~0.3ml of supernatant that was mixed with 0.15ml of 100% ethanol and was added to the spin column step

RNA Quantification

• Diluted samples 1:50 in nucelase-free water
• Used 10µl of sample and 190µl of Qubit Mix
• Ran RNA Quantification Protocol
• Saved samples at -80°C in 2 aliquots (~35µl each)

RNA Concentrations

RNA Quality check of total RNA

• Ran RNA Quality Check Protocol

• Used 1:50 dilution from Qubit above and ran on bioanalyzer
• BioAnalyzer 20160928 Chip

Conclusions

• There is not a substantial increase in RNA quality between the Directzol kit and the RiboPure Kit.
• There still appears to be degradation in some samples.
• Will proceed by extracting all homogenized samples with the Directzol protocol and check quality
• If extracted samples are of poor quality will consider proceeding with siphon only tissue.