qPCR – C.gigas COX1/COX2 Tissue Distribution

Performed qPCR using pooled cDNA from 20110311. Pooled 2uL from each of the following samples groups: Dg 3hr C, Gill 1hr C, Gill 1hr E, Mantle 3hr C, and Muscle 3hr C. Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results). Primers sets run were:

EF1_qPCR_5′,3′ (SR IDs: 309, 310)

Cg_COX1/2_qPCR_F (SR ID: 1192) + Cg_COX1_qPCR_R (SR ID: 1191)- Target = COX1

Cg_COX1/2_qPCR_F (SR ID: 1192) + Cg_COX2_454align1_R (SR ID: 1190) – Target = COX2

Results:

qPCR Report (PDF)

qPCR Data File (CFX96)

Graphs were generated using the BioRad CFX Manager v2.0 software. Expression was normalized to EF1. Also to note, gene efficiency was assumed as 100% by the software since no standard curve was run on the plate. As such, analysis of this data may not be exact.

It’s clear by examining the graphs that the primers being used to differentiate COX1 and COX2 (since they share a common primer: SRID 1192) are differentially expressed. This indicates that the primer sets are indeed amplifying different targets as hoped. This was the primary intention of this qPCR. However, we also now have an idea of tissue distribution of the two genes, as well as their response to V. vulnificus exposre after 1hr. Next step is to perform this qPCR on all the individuals from this experiment as well as the different tissues.

qPCR – C.gigas BB/DH cDNA for PROPS

Performed qPCR using cDNA from 20110311. This was performed for additional reps for TIMP3(BB) (SR IDs:1067 & 1106) and HMGP (SR IDs:359 & 360). Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results).

Results:

qPCR Report (PDF)

qPCR Data File (CFX96)

Will analyze with PCR Miner and incorporate with previous PCR rep done for PROPS with these two genes. Oddly, samples in wells B09 and H09 have weird melt curves. As such, these samples will be excluded from analysis.

Reverse Transcription – C.gigas BB/DH DNased RNA (from 20090507) for PROPS

Performed RT on DNased RNA using Promega MMLV RT and Oligo dT according to manufacturer’s protocol, using 1ug of DNased RNA, but in a 50uL reaction. Due to large number of samples, cDNA was made in PCR plate. Plate layout and calcs are here.

Reverse Transcription – C.gigas DNased RNA (from 20110131) from V.vulnificus Exposure & Tissues (from 20110111)

Performed RT on DNased RNA using Promega MMLV RT and Oligo dT according to manufacturer’s protocol, using 1ug of DNased RNA. Due to large number of samples, cDNA was made in PCR plate. Plate layout and calcs are here. cDNA was diluted 4-fold (to 100uL total volume) based on qPCR done by Emma on 20110202.

SOLiD Sequencing Submission

Submitted the following 8 samples for SOLiD sequencing at HTGU:

 

SB unmeth C.gigas C.gigas gill pool gDNA
SB meth C.gigas C.gigas gill pool gDNA
MA Mercenaria mercenaria gill pool polyA(x2)
BX Mercenaria mercenaria gill pool polyA(x2)
Vt RE22 Vibrio tubiashii (RE22) gDNA
Vt STRAIN Vibrio tubiashii (ATCC 19106) gDNA

 

mRNA Isolation – Pooled Black Abalone Dg RNA (from Abalone Dg Exp 1)

mRNA was isolated for SOLiD sequencing by HTGU. Made two pools of San Nick RNA (Control and Exposed) using equal amounts (5ug) of each individual sample. Individual samples used can be found here. mRNA was isolated using Ambion’s Micro PolyAPurist Kit according to protocol. Procedure was performed two times on each pool and then EtOH precipitated. Samples were resuspended in 10uL of The RNA Storage Solution provided in the kit and spec’d on the Roberts Lab ND1000. Samples were stored @ -80C in the “Next Gen Sequencing Libraries” box.

Results:

Yields are pretty good from both samples (~500ng). However, the OD260/280 values are rather poor.

3’RACE – C.gigas 3’RACE for COX2

Used Cg_COX2_3’RACE_short (SR ID: 1197) & Cg_COX2_3’RACE_long (SR ID: 1196) and the Clonetech SMART RACE cDNA Amplification Kit (unknown acquisition date) to attempt to acquire more 3′ sequence of the C.gigas COX2 isoform. Used Gigas 3’RACE cDNA (from 20080610).

Results:

Gel Loading:

Lane 1: Hyperladder 1

Lane 2: empty

Lane 3: Cg_COX2_3’RACE_long

Lane 4: Cg_COX2_3’RACE_long NTC

Lane 5: empty

Lane 6: Cg_COX2_3’RACE_short

Lane 7: Cg_COX2_3’RACE_short NTC

Lane 8: Hyperladder 1

No products produced. This could be due to a large number of factors. The age of the cDNA (from 20080610) is well beyond what the Clontech manual says for storage term (6 months). Additionally, the Clontech polymerase used was nearly 6 years old. The kit (and its components) are of an unknown age and could factor in to the failure of this procedure. Also, the primers that were designed had less than ideal Tm, per the kit’s recommendations.

May need to sequence some previously purified potential COX2 fragments in order to obtain a more useable region of the gene for RACE.

NanoDrop1000 Comparison – Roberts vs. Young Lab

A previous comparison was performed (see 20110209), but it was determined that the standard DNA being used to test the machines was old/degraded. Lisa ordered a new standard DNA dilutions series (Quant-iT dsDNA Kit; Invitrogen) and these DNAs were used. All DNAs were measured 5 times and were mixed by gently flicking between each measurement. A “blank” was measured between each different [DNA] and, if the reading was > + or – 1ng/uL, the machine was reblanked.

Results:

Quick assessment is that Graham’s NanoDrop1000 is more accurate than ours.

Here is a spreadsheet with averages, standard deviations and experimental error (%). Below are the raw measurements from both machines.

Roberts Lab ND100:

Young Lab ND1000:

qPCR – Check DNased RNA BB01 for Residual gDNA (from earlier today)

Ran qPCR on DNased RNA from earlier today to verify removal of contaminating gDNA. Used C.gigas 18s primers (SR IDs: 156, 157). 0.5uL (~40ng) of DNased RNA was used for testing. This corresponds, roughly, to the amount of sample that would be carried through to qPCR analysis of cDNA, assuming 1ug of RNA was used to make the cDNA (cDNA = 1000ng RNA/25uL = 40ng/uL, 1uL of cDNA in 25uL qPCR reaction). Positive control sample was ~25ng BB16 gDNA (from 20090519). Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results). RNA was stored @ -80C in “Sam’s -80C Box”.

Results:

qPCR Report (PDF)

qPCR Data File (CFX96)

Residual gDNA is present in the sample. So, it’s become apparent that it’s virtually impossible to rid the BB01 RNA of contaminating gDNA. Will discuss with Steven and Mac if it’s feasible to exclude this from the additional PROPS analysis that needs to be done and how this could potentially affect our data. Talked to Steven and, duh, we can just remove the previous BB01 data from our analysis. Will make new batch of cDNA from existing DNased RNA samples.

DNase – C.gigas BB01 (PROPS) RNA (from 20090507)

Since the previous DNase treatment failed for this sample, will repeat but will start with less RNA (5ug instead of 10ug). Need more DNased RNA to finish repeating of PROPS. Some samples had insufficient quantities of DNased RNA remaining in BB01. Used 5ug of RNA and followed Ambion’s “rigorous” protocol, utilizing a total of 2uL of DNAse for each sample. Briefly, samples were incubated @ 37C for 30mins, an additional 1uL of DNase was added to each sample, mixed and incubated for an additional 30mins @ 37C. After finishing protocol, samples were spec’d.

DNase Rxn Calcs:

BB01 (1.824ug/uL): 5ug/1.824ug/uL = 2.74uL RNA + 42.26uL H2O (to 45uL) + 5uL 10X DNase Buffer = 50uL

Results:

RNA looks OK, based on OD260/280. Would like that value to be higher, though. OD260/230 is low, which is typical post-DNased treatment. Will check for residual gDNA via qPCR.