Target Discovery and Validation Reviews and Protocols

In Vivo Assay of Human Angiogenesis 265
3.4.3. Analysis of hPDGFB RNA by Quantitative Reverse Transcription-PCR
1. Amplify human PDGFB-specific transcripts by using 5′-CAT AGA CCG CAC
CAA CGC CAA CTT C-3′ as forward primer and 5′-ATC TTT CTC ACC TGG
ACA GGT CGC AG-3′ as reverse primer at 3.0 mM MgCl 2 and 68°C annealing
temperature.
2. In brief, for hPDGFB determination in the cDNA samples, mix the cDNA (0.5 µL
of the step 1 reaction), dNTPs, primers, MgCl 2 , and enzyme mix containing
SYBR Green to a final volume of 20 µL, load into the glass capillaries, and subsequently
perform quantitative real-time PCR by using LightCycler. Read the fluorescence
intensities at 72°C after each PCR cycle.
3. Quantify hPDGFB copy numbers by using the standard delivered with the PDGF-β
kit from Search-LC.
4. Quantify the human housekeeping gene hydroxymethylbilane synthase by using
the QuantiTect SYBR Green PCR kit at 60°C annealing temperature, 3.5 mM
MgCl 2 concentration with forward primer 5′-GTA ACG GCA ATG CGG CTG-3′
and reverse primer 5′-GGT ACG AGG CTT TCA ATG-3′ as internal control for
the amount of human RNA input.
5. Determine the actual copy numbers of the transcripts in the samples by correlation
between the crossing points of the respective PCRs on test samples and standard
samples by using LightCycler Software 3.
3.5. In Situ Hybridization of hPDGFB on Human Endothelium in Mice
3.5.1. Probe Generation and Labeling
1. A chloramphenicol-resistant pBC vector containing a 1-kb insert for hPDGFB
flanked by upstream EcoRI and downstream NotI was kindly provided by C.
Betsholtz (Department of Medical Biochemistry and Biophysics, Karolinska
Institute, Stockholm, Sweden).
2. Linearize the plasmid by overnight incubation with restriction enzymes EcoRI for
the antisense and NotI for the sense probe. Purify the probes by phenol/chloroform
extraction followed by ethanol precipitation.
3. Transcribe and label the riboprobes by using the DIG RNA labeling kit. Add transcription
buffer, RNase inhibitor, NTP labeling mix, and RNA polymerases T7 or
T3 for the antisense or sense probes, respectively, to the purified templates and
incubate for 2 h at 37°C. Add DNaseI to the probes and incubate for 15 min
(37°C).
4. Rinse the probes by overnight precipitation in ammonium acetate, followed by
ethanol wash twice and resuspension in 10 µg/mL Ribohybe.
5. Prepare the probes for Ventana robot by adjusting the concentration to 0.1 µg/mL
in Ribohybe.
3.5.2. In Situ Hybridization
1. Prepare freshly cut (4-µm) formalin-fixed tissue sections of Matrigel plugs on
SuperFrost glass slides and air-dry at 37°C overnight.