20120501

qPCR - Detection of V.tubiashii Presence and Expression Using VtpA Primers in DNA/cDNA from yesterday

Ran qPCR with VtpA primers on cDNA and DNA (from yesterday) of C.gigas larvae to see levels of V.tubiashii compared to their water filter samples (see 20120326). Master mix calcs are here. Plate layout, cycling params, etc can be seen in the qPCR Report (see Results). Used 1uL of cDNA and 100ng (1uL) of DNA as template.
All samples were run in duplicate.


Results:
qPCR Data File (CFX96)
qPCR Report (PDF)

No detectable levels of expression (or, no expression at all) in any of the cDNA samples.

Below I've put together a very rough comparison of larvae levels, based off of the the standard curve. I have NOT done the full back calculations!! This is data straight out of the qPCR machine, using the standard curve. Due to the large range, I've graphed the data on a logarithmic scale so all the data is visible on the graph.

external image 20120502-r83eaackyu4iasgxta3uicb7kp.jpg


20120430

Dilutions - C.gigas Larval DNA from 20120426

Created dilutions of all samples to 100ng/uL in a volume of 50uL in preparation for qPCR analysis. Calcs are here.


Reverse Transcription - DNased C.gigas Larval RNA from 20120427

Performed reverse transcription using random primers (Promega) diluted 1:100 (5ng/uL) with 175ng of DNased total RNA. Random primers were used because we will be targeting V.tubiashii RNA instead of eukaryotic RNA. Reverse transcription was performed with M-MLV Reverse Transcriptase (Promega) according to the manufacturer's protocol. Calcs are here.



20120427

qPCR - Check DNased RNA from Earlier Today for Residual gDNA

Ran qPCR using V.tubiashii VtpA primers (from Elene; no SR ID). Used 0.5uL of each DNased RNA sample, which equals ~40ng of RNA, which would be the equivalent amount of RNA that would end up in a qPCR rxn after cDNA has been made (using 1uL of cDNA). Used the filter DNA extraction from samples #279 from DATE as a positive control. Master mix calcs are here. Plate layout, cycling params, etc. can be found in the qPCR Report (see Results).

Results:
qPCR Data File (CFX96)
qPCR Report (PDF)

All samples showed up negative, except for the positive control. Will proceed with making cDNA on Monday.

DNase Treatment - C.gigas Larvae RNA from yesterday

Treated 5ug of total RNA (in a 50uL rxn) using Turbo DNA-free (Ambion) according to the "Standard" protocol. Samples were spec'd on the Roberts Lab NanoDrop 1000.

Results:

external image 20120427%20DNAsed%20RNA%20ODs-01.JPG

All samples look good, both quality and quantity-wise. Will check for residual gDNA in these samples via qPCR.




20120426

gDNA Isolation - C.gigas Larvae from Taylor Summer 2011

Samples that had been split from earlier today (see the RNA Isolation below) were resuspended in 1mL of DNAzol (MRC). 100ug of Proteinase K (Fermentas) was added to each sample. Samples were incubated at RT, O/N on a rotator. On 20120427 samples were pelleted 10mins, 10,000g, and supe transferred to fresh tube. DNA was precipitated with 0.5mL of 100% EtOH, mixed gently and pelleted 5mins, 5000g. Supe was discarded, pellets were washed with 1mL 75% EtOH, re-pelleted at same speed as previous step, supe discarded and pellets were resuspended in NanoPure H2O. Samples were spec'd on the Roberts Lab NanoDrop1000.

Results:
Report on the NanoDrop software wouldn't display, so I've entered the concentration of each sample in the table below.
SampleID
ng/uL
201
1364
280
131.7
314
710.3
342
539.4
434
274.4
552
334.8
605
369.7




RNA Isolation - C.gigas Larvae from Taylor Summer 2011

Samples had been stored in RNA Later (Ambion). Samples were pelleted and the RNA Later supe removed. Samples were washed (2x) with 1mL TE (pH = 8.0) to remove excess salt resulting from the RNA Later. Samples were split, roughly equally, into two separate tubes. Samples were pelleted and the supe removed. One tube from each sample was set aside for gDNA isolation using DNAzol (MRC). The other tube was vortexed vigorously in TriReagent (MRC) and the then treated according to protocol. Samples were resuspended in 100uL of 0.1% DEPC-H2O and spec'd on the Roberts Lab NanoDrop 1000.

Results:

external image 20120426%20RNA%20ODs-01.JPG

Overall, the samples look really good. Some samples (280, 434 & 605) required re-specing after the NanoDrop was reblanked in order to get a reading without an error message. They will be DNased and then reverse transcribed.