Tag Archives: Dg

qPCR – Withering Syndrome Phage

Ran qPCR using the following primer sets designed off of the potential WS phage sequence that Stan Langevin has sequenced:

  • 1_ORF25F_225_CSF, 1_ORF25R_399_CSF
  • 2_ORF25_121_CAB, 2_ORF25R_320_CAB
  • 3_ORF20F_121_CSF, 3_ORF20R_326_CSF

Templates tested were abalone digestive gland gDNA:

  • 06:6-41 (4/7/2008) – Positive for withering syndrome and phage; used in the MiSeq run by Stan Langevin.
  • 06:6-53 (4/9/2008) – Positive for withering syndrome and phage; used in the MiSeq run by Stan Langevin.
  • 08:4-1 – Positive for withering syndrome only – no phage.
  • UW08:22-11A – Pinto abalone naive for both withering syndrome and phage.

Master mix calcs are here: 201400820 – qPCR WS phage

All samples were run in duplicate. See qPCR Report (see Results) for plate layout, cycling params etc.

Results:
qPCR Report (PDF): Sam_2014-08-20 14-29-56_CC009827.pdf
qPCR Data File (CFX96): Sam_2014-08-20 14-29-56_CC009827.pcrd

Great news! No amplification in the 08:4-1 (positive for WS, but naive for phage)!! These results strongly suggest that these primers are specific for the WS bacteriophage! This is really cool and exciting. Next steps will be to confirm via in-situ hybridization (ISH).

Additional summary of the results:

Primer set ORF25_CSF shows the highest sensitivity.

Primer set ORF20_CSF fails to amplify anything in 06:6-53

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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.

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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.

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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. :(

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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…

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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.

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