Pam Jensen stopped by and dropped off Tanner crab (Chionoecetes bairdi) hemolymph samples (~300) stored in RNA Later (Ambion). Samples were stored at 4C.
30 gill tissue samples in RNA Later from CA, MA, & MAX each.
30 hemolymph samples (in RNA Later?) from CA, MA, & MAX each.
Presumably these are from the same individuals. Tubes were boxed (a total of 3 boxes), labeled and stored @ -80C.
Here is a note included from Emily with the samples .
Rec’d package of hard clam samples from Emily @ Rutgers on wet ice. Package contained numerous 1.5mL snap cap tubes separated in to groups in zip lock bags. Stored temporarily @ 4C. Will catalog and then store @ -80C.
Three documents included with package:
*Important Note: These were received while I was out of lab. This notebook entry was added 20101021*
Received sets of gill tissue and hemolymph in RNA Later from Rutgers (Emily). Here’s the note that was included with the samples.
Received set of gill tissue in RNA Later MBL (Scott Lindell).
All samples were stored @ -80C.
Rec’d package from Rutgers (Emily Pearson) containing two large Ziplock bags on “wet” ice, each of those containing smaller bags with sample tubes in them. One large bag contains gill tissue samples and the other large bag contains hemolymph samples. Samples will temporarily be stored @ 4C until they can be catalogued and boxed by Lexie later today.
Bled 7 clams from 20090108 and 20090109. Bled clams using a 23g 1.5 needle on a 3mL syringe. Fluid was gathered and ranged from ~0.4-1.0mL. Hemolymph was transferred to individual 1.5mL snap cap tubes and spun @ 100g for 30mins @ 4C. Most of the supe was removed, but left ~100uL in each tube to avoid disturbing any pellet. Samples were stored @ -80C in the red box with previous hard clam hemo samples.
NOTE: One sample was EXTREMELY cloudy. Likely not hemos.
Transferred supe to a fresh tube and added 1mL 70% EtOH to remaining pellet. Spun samples max speed @ 4C 30 mins. Removed supe and washed pellets with 1mL 70% EtOH. Spun max speed 10 mins. Removed supe . Resuspended the “supe” sample in 50uL 0.1%DEPC-H2O and the “pellet” sample in 100uL 0.1%DEPC-H2O.
Results: 260/280 ratios look good. The 260/230 ratios are still horrible. Total yield from these two samples are ~5ug. Will get more hemolymph from clams in order to use more total RNA in the mRNA isolation to maximize cost saving.
Because of the relatively large size of the pellets vs. the amount of RNA, I think another round of precipitation would be best to help remove additional residual salt carryover. Will precipitate O/N according to Ambion PolyA Purist protocol. RNA pellets were resuspended in 250uL of 0.1%DEPC-H2O and precipitated O/N @ -20C.
NOTE: Upon adding 100% EtOH to sample, the solution turned very cloudy and a white precipitate immediately formed inside the tube. I do not think this precipitate is RNA. Tomorrow, before spinning the tube, I will transfer the supe to a fresh tube and process both tubes simultaneously. Hopefully this will remove/eliminate most of the excess salt or whatever seems to be forming the pellet.
Bled 8 clams from 20090108 and 20090109, #4, 6, 8, 15, 16, 17, 21, 26. Bled clams using a 23g 1.5 needle on a 3mL syringe. Fluid was gathered and ranged from ~0.4-1.0mL. Hemolymph was transferred to individual 1.5mL snap cap tubes and spun @ 100g for 30mins @ 4C. Most of the supe was removed, but left ~100uL in each tube to avoid disturbing any pellet. Samples were stored @ -80C in the red box with previous hard clam hemo samples.
Pellets were apparent in all 8 samples, whereas they had not been noticeable before in last week’s bleeds.
Also, 3 clams were found with cracked shells, but alive, including the one pictured below that is split open entirely.
1mL of TriReagent was used to isolate RNA from 3 combined tubes of hemolymph. This resulted in 10 total RNA preps. Pellets were resuspended in 100uL of 0.1% DEPC-H2O and pooled into a single tube and NanoDropped.
Results: RNA solution looked very cloudy and contains a fair amount of insoluble “stuff”. 260/280 ratios also looked bad. Will precipitate O/N according to Ambion PolyA Purist Kit before isolating mRNA tomorrow.