Jan 14, 2018
Our first meeting.
We are completing our RNA extraction. Samples will be sent for sequencing soon !!
Our first meeting.
Practice collection of Salmonberry specimens in Bellevue, WAWe collected the specimens, cleaned them and froze them in dry ice and isopropyl alcohol mix. This stops all cell activity to halt the RNA production and degradation instantly. This can be done by putting the specimens in liquid nitrogen and storing at -80 C. We did not have access to a -80 C freezer so this was a practice run. We were able to collect berries, flower petals, leaves, stem, and root pieces.
Collection at Grass Lawn Park in Redmond, WA: We collected berries, flower petals, leaves, stem and root pieces. We washed them, chopped them, and preserved them in vials with RNALater. RNALater allows storage at -20 C without degradation.
We came across two different types of Salmonberry bushes at Grass Lawn Park, and this could be interesting to look into next spring.
A big thank you to our sponsors who funded the collection materials and RNALater.
Collaboration established with Dr. Madzima: Dr. Thelma Madzima from University of Washington, Bothell graciously volunteered to support our project by providing us the space for our molecular biology research and her expertise in plant molecular biology. Dr. Madzima’s research is currently funded by the UW Bothell, School of STEM.
RNAseq Sponsorship by Covance: Covance Genomics (a division of Labcorp) offered to conduct the RNAseq sequencing in their lab for free of charge. Many thanks to Dr. Kerry Deutsch for arranging this sponsorship.
First Wetlab Work: Our students completed their first trial of RNA extraction with help from Adrianna of Dr. Madzima’s lab. Plant speciments prserved in RNAlater were partially thawed, refrozen in liquid nitrogen and ground into fine powder under liquid nitrogen. Using the fine powder of leaves, stems, petals and red berries, RNA extraction was attempted using the Qiagen Rneasy plant kit.
UV Absorbance values (260/230 and 260/280) were less than ideal for high quality RNA for all tissues tested. Among them, leaves gave the best amount of extracted RNA but the RNA did not meet the quality standards.
A salmonberry plant from Dr. Jae Geller’s backyard was brought to the Madzima lab and its stem, root and leaves were frozen in liquid nitrogen and stored in -80C.
Second Trial of RNA Extraction: In this lab visit, we used the Direct ZOL RNA miniprep Plus kit from Zymo Research to attempt the RNA extraction from the more challenging tissues of root, stem and red berry. The method also incorporated on-column DNAse digestion. Unfortunately this extraction method failed to yield sufficient quantities of RNA from any of the tissues.
Troubleshooting to Improve RNA Quality: Our lab visits in December continued to troubleshoot the RNA extraction method in order to improve absorbance readings 260/230 and 260/280. We repeated RNA extraction procedure with Qiagen and Zymo kits and on-column DNAse digestion using the liq N2 frozen leaf samples (from October) instead of the RNAlater preserved leaf tissues. No improvement in RNA quality was obtained from liq nitrogen frozen tissues. The typical absorbance readings for 260/230 was 0.5-0.6 and 260/280 was 1.1.
First Successful Run of RNA Extraction: We used the trial kit of Plant RNA extraction from Norgen Biotek. The company sent a kit for 4 free trials. Upon processing with this kit, For the first time the liquid nitrogen frozen leaves after processing and extraction yielded high quality RNA 260/230 was 0.9 and 260/280 was 1.6. We repeated the extraction again followed with a further round of RNA clean-up. Absorbance values further improved such that 260/230 was 1.4 and 260/280 was 1.85. The integrity of the purified RNA was also confirmed by gel.