Today I began a big round of RNA isolation, which I will eventually use for QuantSeq libraries and sequence for gene expression. RNA will be isolated using RNAzol, from adult Oly ctenidia tissue after 7 weeks in high/ambient pCo2, and from newly released larvae from parents who had previously been exposed to pCO2 treatments. Larvae are pooled by a daily larval pulse.
Step 1. Homogenize tissues.
I’m homogenizing tissues in liquid nitrogen with mortar + pestle. It’s rather labor intensive, and I lose lots of my tissues, however it’s the best way to fully homogenize, particularly the larvae.
Today I began with ctendia tissues.
- Added 1mL RNAzol to 1.5 mL microcentrifuge tubes.
- Cleaned mortars, pestles, and metal spatulas. Did this by cleaning under hot water, soaking in 10% bleach/DI solution for a minimum of 10 minutes, rinsing thoroughly with DI water, then rinsing with 190 proof ethanol and letting dry.
- Put tubes with RNAzol on scale. Ground tissues to powder, scraped with metal spatula and carefully transferred powder to tube. The goal was 100 mg, but my tissues were mostly too small. I typcially achieved ~20-60 mg.
I did 8 samples at a time (the # of mortar+pestle kits I have), then cleaned and repeated with another 8. Each round of 8 included 2 samples from different cohort + pCO2 treatment. I’m doing the Oyster Bay, Fidalgo Bay and Dabob Bay F1 cohorts.