---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
date: 2015-04-09 04:58:24+00:00
layout: post
slug: rna-isolation-geoduck-foot-in-paraffin-histology-blocks
title: RNA Isolation - Geoduck Gonad in Paraffin Histology Blocks
categories:
  - 2015
  - Protein expression profiles during sexual maturation in Geoduck
tags:
  - Disruptor Genie
  - geoduck
  - gonad
  - histology
  - histology cassettes
  - NanoDrop1000
  - Panopea generosa
  - paraffin
  - PAXgene Tissue RNA Kit
  - RNA isolation
  - RNA quantification
---

Isolated RNA from geoduck gonad previously preserved with the PAXgene Tissue Fixative and Stabilizer and then embedded in paraffin blocks. [See Grace's notebook for full details on samples and preservation](https://genefish.wikispaces.com/Grace%27s+Notebook).



RNA was isolated from only two samples using the PAXgene Tissue RNA Kit (Qiagen) from the following geoduck sample blocks to test out the kit:




    
  * 34

    
  * 42



IMPORTANT:


    
  * Prior to beginning, I prepared Buffer TR1 by adding 10μL of β-mercaptoethanol (β-ME) to 1000μL of Buffer TR1). This will be good for up to six weeks at RT.

    
  * Reconstituted DNase I with 550μL of RNase-free H2O. Aliquoted in 100μL volumes and stored @ -20C in the "-20C Kit Components" box.



Five 5μm sections were taken from each block.

Isolated RNA according to the PAXgene Tissue RNA Kit protocol with the following alterations:


    
  * "Max speed" spins were performed at 19,000g.

    
  * Tissue disruption was performed with the Disruptor Genie @ 45C for 15mins.

    
  * Shaking incubation step was performed with Disruptor Genie

    
  * Samples were eluted with 34μL of Buffer TR4, incubated @ 65C for 5mins, immediately placed on ice and quantified on the Roberts Lab NanoDrop1000.



Samples were stored at -80C in [Shellfish RNA Box #5](https://docs.google.com/spreadsheet/ccc?key=0AmS_90rPaQMzcHdyU1d0MDVMLWpaTWdadnJSd0M4UUE&usp=sharing).

NOTE: The spreadsheet linked indicates other samples exist in the slots that I placed these two samples. Will need to update the spreadsheet to be accurate.

Results:

![](https://eagle.fish.washington.edu/Arabidopsis/20150408%20-%20Geoduck%20block%20RNA%20ODs.JPG)(http://eagle.fish.washington.edu/Arabidopsis/20150408%20-%20Geoduck%20block%20RNA%20ODs.JPG)



![](https://eagle.fish.washington.edu/Arabidopsis/20150408%20-%20Geoduck%20block%20RNA%20ODs%20plots.JPG)(http://eagle.fish.washington.edu/Arabidopsis/20150408%20-%20Geoduck%20block%20RNA%20ODs%20plots.JPG)



Looks like the kit worked! Yields are pretty good (~800ng) from each. The 260/280 ratios are great for both samples. Oddly, the 260/230 ratios for the two samples are pretty much polar opposites of each other; not sure why.

Will proceed with the remainder of the samples that were selected by Steven and Brent. Or, maybe I should try to make some cDNA from these RNA samples to verify the integrity of the RNA...
