Laura’s Notebook: Oly OA RNA isolation – juvenile ctenidia

I’m done with my adult ctenidia & larvae libraries, and have enough kit leftover for ~17 more samples. I’ve decided to prep a few of my juvenile samples, which were collected at the end of the summer deployment. It could be very interesting to assess differences in juveniles and whether they are similar to those observed in the parents that were directly exposed.

I’m doing the whole body samples collected from the Hood Canal and Fidalgo Bay populations after they were deployed in Port Gamble Bay. I have n=4 per population and parental pH treatment (high or ambient). I had wanted to do ctenidia tissues, but then I checked out the frozen samples and noticed that they definitely are not just gill – lots of mantle tissue in there too. Therefore, I decided to do whole-body samples to try to standardize the tissue type.

Step 1: Homogenize tissue (March 6th, 2020)

Need: LN, dry ice, bleach, DI water, mortar + pestle, metal spatulas

  • 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. Added approximately 50 mg.
  • I did 8 samples at a time (the # of mortar+pestle kits I have), then cleaned and repeated with another 8.

Step 2: RNA isolation (March 7th, 2020)

Need: RNAzol, DEPC-treated water, isopropanol, 200-proof ethanol, 1.7 mL tubes

Followed the RNAzol® RT RNA Isolation Reagent protocol for Total RNA isolation, using half of my homogenate, so 500 uL.

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