The 8 hemo samples were pooled and the 4 gonad/d.g. samples were pooled. RNA was isolated. The homo sample was resuspended in 100uL of 0.1%DEPC-H2O and the gonad/d.g. sample was resuspended in 50uL 0.1%DEPC-H2O. RNA was precipitated O/N @ -20C according to Ambion PolyA Purist protocol in preparation for mRNA isolation.
Hemos were collected from the remaining 12 clams as before and stored @ -80C. NOTE: 4 hemocyte samples were extremely cloudy. Possibly not hemos? Maybe gonad/d.g.? Gill tissue was collected from 5 clams and stored @ -80C.
RNA isolated on 20090116 was precipitated over the weekend @ -20C. Samples were treated according to Ambion PolyA Purist protocol and resuspended in 100uL of 0.1% DEPC-H2O. Samples were stored in the red “hard clam” box @ -80C.
Results: 260/280 look good for both gill and hemo samples. 260/230 looks OK for gill, but looks horrible (as usual) for hemos.
RNA was isolated from hard clam gill (0.08g; from 20090109) and from hard clam hemos (from 20090114). One “unknown” sample (from 20090114) was also processed. Pellets were resuspended in 50uL of 0.1%DEPC-H2O. Hemo samples were pooled. Samples were spec’d.
Results: 260/280 looks OK for hemos. The gill sample has excellent 260/280 and pretty good 260/230. Samples will be reprecipitated over the weekend @ -20C according to Ambion PolyA Purist protocol in preparation for mRNA isolation.
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.
Bled 24 hard 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 wiht previous hard clam hemo samples. Clams were numbered and transferred to a holding tank. Gill and mantle tissue was collected from 10 of the clams. The collected tissue and the rest of the carcasses were stored @ -80C.