Ran a full plate for testing well-to-well consistency (or, inconsistency!) of the Opticon 2, since it’s been behaving poorly lately. This will provide us with an idea of whether or not the oddities that we’ve been witnessing have any effect on our actual data.

Used C.gigas gDNA (DH15 from 20100519; 0.5128ug/uL) and IL17 Internal Fw/Rv primers (SR ID: 255, 256), which have previously produced an amplicon with gDNA. Master mix calcs/plate layout/cycling parameters/etc are here.

DNA was combined in master mix so that all wells received ~100ng of gDNA.

Results:

Performed qPCR on Friedman Lab machine targeting immune-related genes in hard clam. Rough plate layout/master mix calcs are here. qPCR report from Friedman Lab machine is here (PDF) and shows cycling params, plate layout and Cts.

Results:

CFX96 Data file is here.

The following primer sets failed to produce an amplicon:

Mm_TRAF6

Mercenaria_Rel

TLR

STI

CytP450-like

Raw fluorescence data was extracted (No baseline subtraction) and processed with PCR Miner. Data workup/analysis is here. Here is a graph of those primer sets producing an amplicon. All were normalized to actin, which exhibited the smallest amount of deviation across all three samples of the normalizing/housekeeping genes analyzed.

As a preliminary run with these genes, there are a number of promising candidates that could yield some interesting data regarding the physiological response of hard clam to exposure to QPX.

qPCR plate layout/setup is here.

Results:

Utilized to sets of primers obtained from the Friedman Lab: VptA (referred to as “Hasegawa”, even though the reference article calls the primers Vtp A) and Vt IGS (referred to as “Lee” primers, presumably from a published article). For template, used “RE22 DNA” that was given to me by Elene. Tube is dated 9/10/09 and has no indication of concentration. Performed qPCR on a set of 10-fold dilutions. Plate layout/qPCR set up is here, along with dilution series used.

Results:

The VptA primer set generated a nice looking set of dilutions with appropriate spacing (~3.2 Ct/10-fold dilution). HOWEVER, the raw fluorescence signal is very low (only 0.4 units; good signal is usually 3-5-fold higher) AND the melting curve doesn’t look that great. The melting curve could look poor due to the low signal, since it doesn’t come up much higher than background levels.

It should be noted that the low fluorescence levels generated could simply be due to the amplicon size generated by these primers. The amplicon size is only 63bp. An amplicon of this size might not be able to incorporate significant amounts of dye to generate a “normal” level of fluorescence (1.25 – 2 units).

The Vt IGS primers failed to generate any product.

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

Results:

qPCR Data File (Opticon): 20100115_113154.tad

Data workup is here: PROPS BB_DH Gene Expression Miner Workup

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

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:

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:

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

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