|
|
Blood, Vol. 113, Issue 19, 4754-4762, May 7, 2009
Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins
Blood O'Connor et al.
113: 4754
Supplemental materials for: O’Connor et al
RT-PCR
The efficiency of the splice-site morpholinos mediated gene knockdown was determined by RT-PCR. Total RNA samples were isolated from control and splice-site morpholino injected embryos using the RNeasy mini kit (Qiagen). For measuring mRNA levels of lrrc32, dcbld2, itga2b, f8, antxr2, bambi, and esam equal amounts of total RNA isolated from two types of embryos were subjected to cDNA synthesis by reverse transcription using One-step RT-PCR kit (Qiagen). Amplification of another cDNA, β-actin, was used as a control. Primer sequences used for the RT-PCR reactions are listed in the Table S1. In order to produce detectable products on gel the following program was used: 50°C 30 minutes, 94°C 15 minutes, 94°C 30 seconds, 58°C 1 minute, 72°C 1.5 minutes (35 cycles), 72°C 5 minutes.
Files in this Data Supplement:
- Document 1. Members of the Bloodomics consortium (PDF, 18.8 KB)
- Table S1. Primers for RT-PCR (PDF, 40.1 KB)
- Table S2. 75 transcripts present in megakaryocytes encoding a protein with a putative transmembrane domain and absent from seven other blood cell types (XLS, 48.5 KB)
- Figure S1. Log2 intensity values for each of the 35 genes encoding transmembrane proteins that are upregulated in MKs compared to all blood cells, but also present in HUVECs (JPG, 81 KB)
-
Genes are ordered by intensity value in MKs and the four selected genes are indicated by arrows.
- Figure S2. Altered splicing of antxr2, bambi, dcbld2, esam, f8, itga2b, and lrrc32 mRNAs in splice MO-injected zebrafish (JPG, 37.6 KB)
-
mRNA levels of antxr2, bambi, dcbld2, esam, f8, itga2b, and lrrc32 in control (−) and MO-injected 3dpf larvae (+) were measured by RT-PCR, from equal amounts of total RNA. Amplification of β-actin, was used as a control. The 1 kb marker band is indicted by an arrow.
- Video 1. Thrombus formation in CD41-GFP transgenic fish in a laser-induced arterial injury model (WMV, 0.99 MB)
-
The caudal artery (CA) of 4 dpf CD41-GFP transgenic zebrafish was injured using an ablating laser and thrombus formation over 1 minute shown. The site of injury is indicated by a star. Direction of blood flow is shown by an arrow. CV: caudal vein. The thrombocytes appear as circulating bright cells.
|
|
