RT-PCR analysis for R01 submission 6/6/16¶

051816: Monique has catalogued all of Carola's RNA samples, from fall 2015 ("RTsamples" below). We will reverse-transcribe n=4 WT, HET, and HOMO granulosa cell (GC) RNA samples, and will perform qPCR using the below primer sets.

Monique: Can we find the concentrations for these samples in Carola's notebook(s)? If so, can you please add to your .csv file? - JJ

The two hypotheses that we're following up with are: 1) precocious differentiation occurs in FXPM granulosa cells as seen in elevated levels of cumulus and mural gene expression 24h post-PMSG. A nice Christenson review

2) decreased expression of anti-apoptosis genes results in increased GC death.

Primers ordered 051816 from realtimeprimers.com¶

Name	Product Number
Lars2	VMPS-3445
Tnfaip6	VMPS-6720
Has2	VMPS-2725
Egr1	VMPS-1863
Btg2	VMPS-725
Btg1	VMPS-724
Ptgs2	VMPS-5166
Mouse Housekeeping Gene	MHK-1

Additional primers ordered 051816 from IDT¶

Name
Pgr
Naip7

RTsamples <- read.csv("Carola_RNA_Inventory.csv")
RTsamples

Mitochondrial isolation kit and activity assays for R01 submission 6/6/16¶

Tobias Holden has been invited to help perform these assays. We anticipate needing to do granulosa cells from stimulated (24 hours post-PMSG) and unstimulated animals. Citrate synthase assay is the top priority, followed by membrane potential.

Activity Assays ordered 051816:¶

Name	Supplier	Product Number
MitoCheck® Mitochondrial (Tissue) Isolation Kit	Cayman Chemical	701010-1EA
MitoCheck Citrate Synthase Assay	Cayman Chemical	701040
Mitochondrial Membrane Potential Assay	Abcam	ab113850

	cell_type	genotype	date
1	MII	WT	1-Oct
2	MII	HET	1-Oct
3	MII	HOMO	1-Oct
4	MII	WT	30-Sep
5	MII	HET	30-Sep
6	MII	HOMO	30-Sep
7	MII	WT	24-Sep
8	MII	HET	24-Sep
9	MII	HOMO	24-Sep
10	MII	WT	22-Sep
11	MII	HET	22-Sep
12	MII	HOMO	22-Sep
13	MII	WT	10-Sep
14	MII	HET	8-Sep
15	MII	HOMO	17-Sep
16	GC	WT	1-Oct
17	GC	HET	1-Oct
18	GC	HOMO	1-Oct
19	GC	WT	30-Sep
20	GC	HET	30-Sep
21	GC	HOMO	30-Sep
22	GC	WT	24-Sep
23	GC	HET	24-Sep
24	GC	HOMO	24-Sep
25	GC	WT	22-Sep
26	GC	HET	22-Sep
27	GC	HOMO	22-Sep
28	GC	WT	15-Sep
29	GC	HET	15-Sep