Category Archives: PROPS

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

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

 

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.

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.

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.

qPCR – Mac’s gigas DNased RNA from earlier today

Performed qPCR on the DNased oyster RNA from earlier today with Gigas_18s_F/R primers to verify removal of detectable gDNA in the samples, since the qPCR from 20090508 indicated residual gDNA was still present in some of the DNase treated RNA. Plate layout/set up is here.

Results: Samples BB# 2, 5, 6, 15 still came up as positive for gDNA contamination. These will NOT be used to make cDNA for subsequent qPCRs.

DNase Treatment (Rigorous!) – Mac’s gigas RNA/Re-DNased RNA from 20090507 & 20090508, respectively

Followed the rigorous protocol for Ambion’s Turbo DNA-free protocol for the following RNAs:

BB#11-20

DH#11-20

Used 10ug of RNA (200ng/uL) in 50uL as directed. Here are the calcs for FF and DH.

Followed standard protocol on DNased samples from 20090508:

BB#1-10

DH#1-10

The standard protocol should be fine for these samples, since the procedure worked on Friday for some of them.

DNA Isolation – Mac’s gigas samples from 20090505 & 20090506

Isolated gDNA according to Molecular Research Center TriReagent protocol from BB#1-20 and DH#1-20. Resuspended DNA in 600uL of 8mM NaOH. Spec.

Results: HORRIBLE! This is some of the worst “DNA” I’ve ever seen. Peaks everywhere EXCEPT at 260nm. Here’s a link to the actual numbers. It’s a text file and is comma separated, so you should open with Excel for it to be readable.

Spoke with Steven. Will pursue RNA instead of continuing down this path for now.

qPCR – Re-DNased oyster RNA from today

Performed qPCR on the re-DNased oyster RNA from earlier today with Gigas_18s_F/R primers to verify removal of detectable gDNA in the samples, since the initial qPCR from yesterday indicated residual gDNA was still present in the DNase treated RNA. Plate layout/set up can be found here.

Results: About 4 samples in each site set are NEGATIVE for gDNA. That means the remainder still have detectable levels of gDNA. Boo.