RNA was isolated in 1mL TriReagent, according to protocol. Samples were resuspended in 50uL 0.1% DEPC-H2O and spec’d. RNA was stored @ -80C in “Shellfish RNA Box #4“.
Results:
All gill RNA looks nearly perfect (based on 260/280 and 260/230 values). Muscle RNA is only OK (based on 260/280 and 260/230 values).
Used COX primers (SR IDs 1060, 1061) and cDNA from 20080327, which consisted of 7 control gigas gill and 7 vibrio-exposed (24hrs) gigas gill samples, labeled as C# and VE#, respectively. The experiment was a 24hr. exposure live Vibrio vulnificus, parahaemolyticus Cf = 2.055×10^11 (6.85×10^7 Vibrio cells/oyster).
Note: Used a free sample of 2x Brilliant III Ultra Fast SYBR Green QPCR Master Mix (Stratagene) for this qPCR. Mixed components and set up cycling params according to the manufacturer’s recommendation for the BioRad CFX96.
Master mix calcs are here. Plate layout, cycling params, etc. can be see in the qPCR Report (see Results).
Results:
PCR Miner analysis is here. There appears to be an increase in COX expression in samples exposed to Vibrio sp. (see graph below), however, I have not determined if the results are significant.
Used new cyclooxygenase primers (SR IDs 1060, 1061) to see how they performed and to evaluate tissue distribution. Tissue distribution was evaluated using the following cDNAs made on 10/27/10 from Emma:
Gigas Digestive Gland
Gigas Gill
Gigas Mantle
Gigas Muscle
qPCR Master Mix calcs are here. Plate layout, cycling parameters, etc can be found in the qPCR Report (see Results).
Results:
qPCR Report (PDF).
Amplification is present in all four tissue types and the melting curve looks good. So, these primers are good to go. Steven suggests checking to see if we see a change in gene expression from an old experiment of Gigas exposed to high levels of Vibrio tubiashii. Will round up some old cDNA for this.
1st of 2 rounds of digests were performed. Calculations are here. Samples were incubated 37C for 3hrs, heat inactivated @ 80C for 30mins and then stored @ -20C.
Continued with 2nd round of digestions from yesterday. All samples were resuspended in 25uL of H2O yesterday, so brought volume up to 44uL with H2O, added 5uL of appropriate 10X Buffer (HpaII = NEB Buffer #4, MspI = NEB Buffer #1), added 1uL of enzyme, incubated 37C for 3hrs. Heat-inactivated all samples @ -80C for 30 mins.
Restriction digests were mixed with equal volume (50uL) of phenol:chloroform:IAA (25:24:1) and centrifuged 16,000g for 5mins @ 4C. Aqueous phase was transferred to a clean tube and an equal volume (50uL) of chloroform was added. Samples were mixed and centrifuged 16,000g for 5mins @ 4C. Aqueous phase was transferred to clean tubes and stored @ -20C. Will EtOH precipitate and spec on Monday.
Restriction digests from yesterday were mixed with equal volume (50uL) of phenol:chloroform:IAA (25:24:1) and centrifuged 16,000g for 5mins @ 4C. Aqueous phase was transferred to a clean tube and an equal volume (50uL) of chloroform was added. Samples were mixed and centrifuged 16,000g for 5mins @ 4C. Aqueous phase was transferred to clean tubes and EtOH precipitated, according to protocol. Samples were resuspended in 25uL of H2O and spec’d.
Samples are labeled as Parent (P), #, tissue, enzyme (MspI = M, HpaII = H, Undigested = U)
Results:
Here is a link to a spreadsheet with ODs. Average recovery was ~734ng, which is only a 36% recovery (started with 2000ng). Will need to discuss with Mac and Steven to see if it’s worth continuing with these sample through a second round of digests/phenol:chloroform extraction/EtOH precipitation, as I don’t know what quantity of DNA is required/desired for the subsequent methylation specific PCR (MSP), OR if I should/need to perform a repeat of these 1st-round digestions in order to end up with sufficient DNA for MSP.
Set up restriction digests for subsequent analysis by methylation specific PCR (MSP). This will be the first of two rounds of digestion with the same enzyme on each sample. Samples and master mixes are here. Samples were incubated 3hr. @ 37C. All samples were heat inactivated at 80C for 30mins and then stored @ -20C.
Received 82 gill samples in RNA Later in 3 microfuge tube racks from MBL (Scott Lindell). Samples were catalogued, boxed (1 box) and stored at -80C.
The live clams were received in two bags one containing ~12 labelled MA4 and one containing ~12 labelled BX1. Clams were NOT counted and the quantity may be different. Clams were temporarily stored @ 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 .
