Tag Archives: DH

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 – BB & DH cDNA (from yesterday)

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

EW778094 (“Ficolin 3″) –

AJ422120.p.cg.6 (“MDR49″, “Multidrug resistance protein homolog 49″) – This was upregulated in DH SOLiD data.

qPCR set up and plate layout can be found here.

Results:

qPCR Data File (Opticon): 20091224_092959.tad

Data workup: PROPS BB_DH Gene Expression Miner Workup

Preliminary results show that the EW778094 primer set looks bad; bad fluorescence profile and poor melt curves. Primers may require optimization.

 

 

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

AM855874.p.cg.6 (“CP17A”, “Steroid 17-alpha-hydroxylase/17″) – This was upregulated in DH SOLiD data.

AM857078.p.cg.6 (“C1QT4″, Complement C1q tumor necrosis factor-related protein 4″) – This was upregulated in DH SOLiD data.

qPCR set up and plate layout can be found here.

Results:

qPCR Data File (Opticon): 20091224_125230.tad

Data workup: PROPS BB_DH Gene Expression Miner Workup

 

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Reverse Transcription – BB & DH DNased RNA (from 20090514)

Made a fresh, double batch (50uL rxn instead of 25uL) of cDNA according to Promega MMLV protocol using oligo dT primers. cDNA was put into a plate for faster qPCR loading. cDNA calcs and plate layout are here. Briefly, RNA and oligo dTs were combined, brought up to 37uL, heated @ 70C for 5mins and immediately placed on ice. RT master mix was made (RT master mix calcs are here), 13uL was distributed to each well. Samples were incubated @ 42C for 1hr and then 95C for 5mins.

UPDATE: cDNA plate was discarded 20120320 by SJW.

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mRNA Isolation – Gigas BB and DH samples previously treated with Ribominus Kit (by Mac)

Was given ~0.5ug of each of these two RNA samples and processed them with Ambion’s microPolyA Purist Kit according to protocol. After elution, the samples were EtOH precipitated @ -80C for 30mins, pelleted 30mins 16,000g for 30mins, 4C. Supe removed, RNA washed with 1mL 70% EtOH and spun 10mins 16,000g, 4C. Supe removed. Resusupended in 10uL of The RNA Storage Solution and gave back to Mac.

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mRNA Isolation – Gigas BB and DH samples previously treated with Ribominus Kit (by Mac)

Was given 1ug of each of these two RNA samples and processed them with Promega’s PolyA Tract Kit according to protocol. After elution, the samples were EtOH precipitated @ -20C for 30mins, pelleted 30mins 16,000g for 30mins, 4C. Supe removed, RNA washed with 1mL 70% EtOH and spun 15mins 16,000g, 4C. Supe removed. Resusupended in 15uL of 0.1% DEPC-H2O and spec’d.

Results: No measurable amount of RNA in either sample. Samples were stored @ -80C.

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DNA Methylation Test – Gigas site gDNA (BB & DH) from 20090515

Used BB & DH samples #11-17 for procedure. Followed Epigentek’s protocol. My calcs for dilutions/solutions needed are here. All solutions were made fresh before using, except for Diluted GU1 which was made at the beginning of the procedure and stored on ice in a 50mL conical wrapped in aluminum foil. Used 100ng total (50ng/uL) of each sample gDNA. No standards for a standard curve based on speaking with Mac.

WELL SAMPLE WELL SAMPLE
A01 BB11 A02 DH11
B01 BB12 B02 DH12
C01 BB13 C02 DH13
D01 BB14 D02 DH14
E01 BB15 E02 DH15
F01 BB16 F02 DH16
G01 BB17 G02 DH17
H01 Pos. Control H02 Blank

Results: Above is the graph of the results. Although it’s only a small difference between the two sites, it is statistically significant. The calcs for this graph can be found here (Excel file). It should be noted that this graph was generated using estimated values from the standard curve provided in the manufacturer’s protocol. This was done because 1) I did not run standards to generate my own curve and 2) calculating the “% methylation” not using the formula that utilizes the standard curve was giving ridiculously high values (e.g. 350%).

Here is the raw data generated by the plate reader for a 1s read (Excel file) and a 0.1s (Excel file) read. Both reads have nearly identical values.

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gDNA Isolation – Mac’s BB and DH site samples

Due to failure of gDNA isolation via the TriReagent method (see 20090511) used Qiagen DNeasy Kit. Digested samples for 3 hrs. at 55C in Proteinase K according to protocol. Performed on a subset of each site samples: BB#11-18 & DH#11-18.

Results: Excellent yields and superb quality.

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Reverse Transcription – Mac’s gigas DNased RNA from 20090512

Performed RT using Promega M-MLV RT according to M-MLV protocol and used 0.5ug oligo dT per ug of RNA on all BB and DH site samples that were negative for gDNA (see qPCR results 20090512). Calculations and work up are here. Samples were set up in a plate to facilitate sample loading in subsequent qPCRs.

UPDATE: cDNA plate was discarded 20120320 by SJW.

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