Dataset Information

LC-MS/MS profiling of sample processing optimization of formalin-fixed, paraffin embedded tissues

ABSTRACT: Despite a clinical, economic, and regulatory imperative to develop companion diagnostics, precious few new biomarkers have been successfully translated into clinical use, due in part to inadequate protein assay technologies to support large-scale testing of hundreds of candidate biomarkers in formalin-fixed paraffin embedded (FFPE) tissues. While the feasibility of using targeted, multiple reaction monitoring mass spectrometry (MRM-MS) for quantitative analyses of FFPE tissues has been demonstrated, protocols have not been systematically optimized for robust quantification across a large number of analytes, nor has the performance of immuno-MRM been evaluated. To address this gap, we used a test battery approach coupled with MRM-MS (targeting 515 analytes) to evaluate quantitatively the performance of three extraction protocols in combination with three trypsin digestion protocols (9 processes). An optimum process based on RapiGest buffer extraction and urea-based digestion was identified. The optimized process is generally amenable and enables highly reproducible, multiplex, standardizable quantitative MRM in archival specimens.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Breast Cancer Cell

DISEASE(S): Breast Cancer

SUBMITTER: Jacob Kennedy

LAB HEAD: Paulovich, Amanda G

PROVIDER: PXD003770 | Pride | 2016-07-25

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	FFPE_Aim1a_LT_RG_D1_063015_01.raw	Raw
	FFPE_Aim1a_LT_RG_D2_070115_01.raw	Raw
	FFPE_Aim1a_LT_RG_D3_070215_01.raw	Raw
	FFPE_Aim1a_LT_TFE_D1_063015_01.raw	Raw
	FFPE_Aim1a_LT_TFE_D2_070115_01.raw	Raw

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Project description:Background: The horn fly, Haematobia irritans (L.), is an obligate blood-feeding parasite of cattle and control of this pest is a continuing problem in the United States and other parts of the world. Worldwide annual economic losses attributable to this pest surpass $1 billion. The fly is becoming resistant to pesticides and new control technologies are needed by cattle producers. Results: Dominant conditional lethal gene systems are being investigated as population control technologies against agricultural insect pests. One of the critical components of these systems is a highly expressed female-specific gene promoter which can be used to drive expression of a lethality-inducing gene. To identify candidate genes to supply this gene promoter, microarrays were designed from a recently developed horn fly EST database and probed to identify female-specific and larval-specific differential gene expression. Analysis of dyeswap experiments found 432 and 417 transcripts which were over- and under-expressed in adult female flies, respectively, compared to adult male flies. Additionally, 419 and 871 transcripts were over- and under-expressed in first instar larvae compared to adult flies. Three transcripts were identified which were over-expressed in adult females flies compared to adult males and which also were over-expressed in the first instar larval lifestage compared to adult flies. Conclusion: We have identified 3 strong candidates for further evaluation as a gene promoter source for the development of a female-specific conditional lethality system in the horn fly. One of these candidates, the putative nanos orthologue, has a high female-to-male expression ratio, has a moderate expression level in first instar larvae, and has been well characterized in D. melanogaster. Further investigations leading from this microarray analysis will include transformation of the horn fly and evaluation of the female conditional lethal system components for applicability to the specific biological parameters of natural populations of the horn fly. Keywords: evaluation of 1st instar larvae genes vs pooled adult + egg genes In short, RNA from female horn flies was labeled and hybridized against labeled RNA from male horn flies in a dyeswap design. Similarly, RNA from first instar larvae was labeled and hybridized against labeled a 50:50 mix of RNA from adult male and female flies. All labeling, hybridization and washing procedures were performed according to respective manufacturer protocols. For each sample, 10 ïg of total RNA and 50-fold dilution of Agilent two color spike-in control RNA kit (Agilent Technologies Inc.) were labeled with either CyDye3-dCTP or CyDye5-dCTP (Amersham Biosciences, Piscataway, NJ) using the LabelStar kit (Qiagen Inc., Valencia, CA) and oligo-dT and Random nonamers (Sigma-Aldrich Inc., St. Louis, MO). Labeled cDNA was hybridized to the Agilent microarrays using the Gene Expression Hybridization kit (Agilent Technologies Inc.) following manufacturerâs protocols. Arrays were washed with Gene Expression Wash Buffer kit (Agilent Technologies Inc.) A total of 8 arrays, including a dye swap for each RNA replicate (4 biological replicates), were analyzed to obtain genes that were consistently regulated while limiting false discover rate (FDR) below 2.5% [26].

Project description:Background: The horn fly, Haematobia irritans (L.), is an obligate blood-feeding parasite of cattle and control of this pest is a continuing problem in the United States and other parts of the world. Worldwide annual economic losses attributable to this pest surpass $1 billion. The fly is becoming resistant to pesticides and new control technologies are needed by cattle producers. Results: Dominant conditional lethal gene systems are being investigated as population control technologies against agricultural insect pests. One of the critical components of these systems is a highly expressed female-specific gene promoter which can be used to drive expression of a lethality-inducing gene. To identify candidate genes to supply this gene promoter, microarrays were designed from a recently developed horn fly EST database and probed to identify female-specific and larval-specific differential gene expression. Analysis of dyeswap experiments found 432 and 417 transcripts which were over- and under-expressed in adult female flies, respectively, compared to adult male flies. Additionally, 419 and 871 transcripts were over- and under-expressed in first instar larvae compared to adult flies. Three transcripts were identified which were over-expressed in adult females flies compared to adult males and which also were over-expressed in the first instar larval lifestage compared to adult flies. Conclusion: We have identified 3 strong candidates for further evaluation as a gene promoter source for the development of a female-specific conditional lethality system in the horn fly. One of these candidates, the putative nanos orthologue, has a high female-to-male expression ratio, has a moderate expression level in first instar larvae, and has been well characterized in D. melanogaster. Further investigations leading from this microarray analysis will include transformation of the horn fly and evaluation of the female conditional lethal system components for applicability to the specific biological parameters of natural populations of the horn fly. Keywords: adult male vs female hornfly gene expression In short, RNA from female horn flies was labeled and hybridized against labeled RNA from male horn flies in a dyeswap design. Similarly, RNA from first instar larvae was labeled and hybridized against labeled a 50:50 mix of RNA from adult male and female flies. All labeling, hybridization and washing procedures were performed according to respective manufacturer protocols. For each sample, 10 ïg of total RNA and 50-fold dilution of Agilent two color spike-in control RNA kit (Agilent Technologies Inc.) were labeled with either CyDye3-dCTP or CyDye5-dCTP (Amersham Biosciences, Piscataway, NJ) using the LabelStar kit (Qiagen Inc., Valencia, CA) and oligo-dT and Random nonamers (Sigma-Aldrich Inc., St. Louis, MO). Labeled cDNA was hybridized to the Agilent microarrays using the Gene Expression Hybridization kit (Agilent Technologies Inc.) following manufacturerâs protocols. Arrays were washed with Gene Expression Wash Buffer kit (Agilent Technologies Inc.) A total of 8 arrays, including a dye swap for each RNA replicate (4 biological replicates), were analyzed to obtain genes that were consistently regulated while limiting false discover rate (FDR) below 2.5% [26].

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LC-MS/MS profiling of sample processing optimization of formalin-fixed, paraffin embedded tissues

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