Tag Archives: Immomix

qPCRs – Mac’s BB/DH cDNA from 20091223

GNRR2 and CALL primer sets used. These are duplicates based on initial differences seen between BB and DH expression. qPCR set up and plate layout is here.

Results:

qPCR Data File (Opticon): 20100111_180230.tad

Data workup: PROPS BB_DH Gene Expression Miner Workup

 

SPI and CP17A primer sets used. These are duplicates based on initial differences seen between BB and DH expression. qPCR set up and plate layout is here.

Results:

qPCR Data File (Opticon): 20100111_141711.tad

Data workup: PROPS BB_DH Gene Expression Miner Workup

 

EF1 primers in duplicate, since I had not done these primers yet. qPCR set up and plate layout is here.

Results:

qPCR Data File (Opticon): 20100111_102001.tad

Data workup: PROPS BB_DH Gene Expression Miner Workup

 

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qPCRs – Tim’s Adult Gigas gill cDNA (from 20091009)

Duplicates of earlier qPCRs.

Primers: Cg_P450 and TNFRAF_5’/3′.

qPCR set up and plate layout are here.

Results:

 

Duplicates of earlier qPCRs.

Primers: Cg_IkB_F997, R1213 and Cg_Prx6_F270, R439.

qPCR set up and plate layout can be found here.

Results:

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qPCRs – Tim’s Adult Gigas gill cDNA (from 20091009)

Duplicates of earlier qPCRs.

Primers: Cg_HIF1 and IL17 Iso D.

The previous version of IL17 primers used (IL17 internal) were NOT the ones used for the paper. The IL17 Iso D are the correct primers and are the same ones that Tim previously used on the juvenille gill samples.

qPCR set up and plate layout can be found here.

Results:

Duplicates of earlier qPCRs.

Primers: EF1 and IL17 Iso D.

The previous version of IL17 primers used (IL17 internal) were NOT the ones used for the paper. The IL17 Iso D are the correct primers and are the same ones that Tim previously used on the juvenille gill samples. qPCR set up and plate layout can be found here.

Results:

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qPCR – BB & DH cDNA (from 20091223)

qPCR was set up on these cDNAs using the following primers:

FP010108.p.cg.6 (“DJB12″, “DnaJ homolog subfamily B member 12″) – This was upregulated in DH SOLiD data.

AJ565670.p.cg.6 (“TOP1″, “DNA topoisomerase 1″) – This was upregulated in BB SOLiD data.

qPCR set up and plate layout can be found here.

Results:

qPCR Data File (Opticon): 20091229_164912.tad

Data workup: PROPS BB_DH Gene Expression Miner Workup

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qPCRs – BB & DH cDNA (from 20091223)

qPCR was set up on these cDNAs using the following primers:

AM861391.p.cg.6 (“BDEF”, “Big Defensin”) – This was upregulated in DH SOLiD data.

AM904566.p.cg.6 (“GNRR2″, “Gonadotropin-releasing hormone II receptor”) – This was upregulated in DH SOLiD data.

qPCR set up and plate layout can be found here.

Results:

qPCR Data File (Opticon): 20091228_102019.tad

Data workup: PROPS BB_DH Gene Expression Miner Workup

 

 

qPCR was set up on these cDNAs using the following primers:

CU988730.p.cg.6 (“TIMP3″, “Metalloprotease inhibitor 3″) – This was upregulated in DH SOLiD data.

CU990442.p.cg.6 (“CALL”, “Calmodulin-like protein”) – This was upregulated in DH SOLiD data.

qPCR set up and plate layout can be found here.

Results:

qPCR Data File (Opticon): 20091228_135507.tad

Data workup: PROPS BB_DH Gene Expression Miner Workup

 

 

qPCR was set up on these cDNAs using the following primers:

CU994646.p.cg.6 (“CATL”, “Cathepsin L”) – This was upregulated in DH SOLiD data.

ES789598.p.cg.6 (“GSTA”, “Glutathione S-transferase A”) – This was upregulated in DH SOLiD data.

qPCR set up and plate layout can be found here.

Results:

qPCR Data File (Opticon): 20091228_165801.tad

Data workup: PROPS BB_DH Gene Expression Miner Workup

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qPCRs – Tim’s adult gigas challenge cDNA (from 20091009)

Set up qPCR with Cg_P450 primers and TNFRAF3’/5′ primers. Plate layout/setup is here.

Results: Processed with PCR Miner. Normalized to EF1. Standard Error bars. Here is spreadsheet with workup.

P450: The two control samples (AC & CC) show significant difference in expression between the Air and CO2 treated samples. The Vibrio treated samples (AV & CV) show no difference in expression between Air and CO2 treatments.

The Air samples (AC & AV) show significant difference in expression between the Control (AC) and Vibrio treated (AV) samples.

TNFRAF3: Expression levels of the Vibrio treated samples (AV &CV) were too low for Miner processing.

There are significant differences in expression between the Air (AC) and CO2 treated (CC) samples.

 

Set up qPCR with Cg_IkB primers and Cg_Prx6 primers. Plate layout/setup is here.

Results: Processed with PCR Miner. Normalized to EF1. Standard Error bars. Here is spreadsheet with workup.

IkB:

No difference in expression between Air (AC & AV) and CO2 (CC & CV) treatments.

Appears to be a significant difference between the Air Control (AC) and the Air Vibrio treated (AV) samples.

Prx6:

Significant difference in expression between Air Control (AC) and CO2 Control (CC) samples, but no difference in the Vibrio treated samples.

Significant difference in expression between the Air Control (AC) and the Air Vibrio treated (AV) samples, but no difference in the CO2 treated Vibrio samples (CC & CV).

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qPCR – Tim’s adult gigas challenge cDNA (from 20091009)

Set up qPCR with Cg_HIF1 (hypoxia induced factor 1) primers and prostaglandin E2 primers. Plate layout/setup is here.

Results: Processed with PCR Miner. Normalized to EF1. Standard Error bars. Here is spreadsheet with workup.

HIF1:

No significant differences between any treatments.

Prostaglandin E2:

No significant differences between any treatments.

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qPCR – Tim’s adult gigas challenge cDNA (from today)

Set up qPCR with EF1 primers and IL17 Internal primers. Plate layout/setup is here. Note: gDNA sample used as a “positive” control will NOT amplify with the EF1 primers.

Results: Processed with PCR Miner. Normalized to EF1. Standard Error bars. Here is spreadsheet with workup.

IL17 Internal:

No significant differences between any treatments.

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qPCR – Tim’s adults gigas challenge re-DNased RNA (from today)

Performed qPCR using q18s primers on re-DNased RNA (1:100 dilution to match final concentration of template after making cDNA). qPCR set up and plate layout are here.

gDNA dilutions were used as positive controls. gDNA = BB11 (0.49ug/uL) from 20090519. Used 5uL of 1:10, 1:100 and 1:000 dilutions.

Results: re-DNase-ing the samples seems to have worked. Positive controls are the only samples to come up. Will proceed to making cDNA.

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