Tag Archives: digestive gland

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


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.

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.


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

DNase – DNase C.gigas RNA from 20110120, 20110121 and 20110124

5ug of RNA was DNased using Ambion’s Turbo DNA-free kit, following the rigorous protocol (0.5uL of DNase for 30 mins then additional 0.5uL of DNase for 30mins). Calcs for DNase reactions are here. RNA was stored @ -80C in Shellfish RNA Boxes 4 and 5. Samples will be spec’d on Monday.


Overall, the RNA looks really good (based on OD 260/280 numbers). Not surprisingly, the OD 260/230 values for all samples dropped, likely due to the addition of the buffer (salts) used in the DNase reaction. Emma says she will check these samples for residual DNA.

–UPDATE (20110131)– Emma checked all DNased RNA samples on 20110131 using C.gigas 18s primers (SR ID: ?). She has not listed the results of the whether or not all samples are clean or if some still have residual gDNA carryover.

–UDPATE (20110201– Samples that appear to have residual gDNA carryover based on Emma’s qPCR on 20110131: Muscle C6, Gill 1hr C2 & E2.

RNA Isolation – Various C.gigas Tissue from 20110111

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


RNA looks OK. Not surprising, but mantle and Dg/Gonad tissues ended up with poor OD260/230 ratios. This has been observed in the past with these tissue types.

qPCR – COX qPCR Primer Test and Tissue Distribution

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).


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.

qPCR – DNased Abalone Dg RNA from 20090625

Ran qPCR on gDNA (06:50-10) to test new primers (H.iris_actin_intron_Fw/Rv) designed to bind only to a region in an intron of the H.iris actin gene. Hopefully there’s enough homology between H.iris (primer source) and H.cracherodii (template source) for this to work. PCR setup/plate layout is here. Anneal temp 50C.

Results: No signal. :(

qPCR – Abalone Dg DNased RNA from yesterday and earlier today

Check Abalone RNA for residual gDNA contamination after DNase treatment. Ran qPCR with H.crach_16s_SYB_F/R primers. Plate layout/PCR set up here.

Results: Still gDNA in virtually every sample! This is totally insane. Will find/design primers that will only amplify gDNA to aid in subsequent analysis…

EtOH Precipitation – DNased Abalone Dg RNA from yesterday AND the 07:12 set (DNased by Lisa 20090306)

Due to the excessive number of times that these samples have been DNased, I’m concerned that the buffer is becoming too concentrated. So, I’m performing an EtOH precip on them to clean them up and then proceed with another round of DNase treatment.

0.1 volumes of 3M sodium acetate (pH = 5.2) were added to each sample. Then, 2 volumes of 100%, ice-cold EtOH were added. Samples were mixed and stored @ -20C for 2hrs. Samples were then centrifuged @ 4C, 16,000g for 30mins. Supe removed. Added 1mL of 70% EtOH and then centrifuged samples @ 4C, 16,000g for 15mins. Supe was removed, tubes were spun briefly to pool residual EtOH and the remaining EtOH was removed. RNA was resuspended in 20uL of 0.1% DEPC-H2O and spec’d.

Results: The following samples appear to have no RNA after precipitation:

06:5-34, 37; 06:6-44, 45, 46, 47, 52; 08:4-9, 10, 13, 15-18; All of the 07:12 samples.

Since I did not DNase the 07:12 samples, I can’t say whether or not this result was expected (e.g. due to low initial concentration). The others that have no remaining RNA are a surprise and is a bit disconcerting.

Will take fresh RNA aliquots of those samples for DNasing. For those samples still having RNA post-recip, I will just use them as they are, as most concentrations are in the recommended range for DNasing, according to the Ambion Tubro DNA-free kit.