Target Discovery and Validation Reviews and Protocols

Pancreatic Cancer 77
11. Incubate overnight at 37°C. Keep the rest of the transformation mixtures at 4°C.
Plasmids with insert should have hundreds to thousands of colonies, whereas control
plates should have zero to tens of colonies.
12. Check the insert sizes by performing colony-PCR. Check at least 36 colonies of
each ligation to calculate the average insert size. Set up 25-µL PCR reactions by
using the M13-forward and M13-reverse primers. Use a sterile pipet tip, or a
toothpick, to pick colonies. Suggested parameters for PCR are as follows: (A)
1cycle:95°C for 2 min, (B) 30 cycles: 95°C for 30 s, 56°C for 1 min, 70°C for
1 min, (C) 1 cycle: 70°C for 5 min.
13. Electrophorese 5 µL of sample on a 1.5% agarose TAE gel at 100 V until the purple
bromophenol blue band is near the bottom of the gel. Load a 1-kb and 100-bp
ladder to determine the insert size. Place 1 µL of 10X loading buffer in the wells
of a 96-well PCR plate. Transfer 5 µL of the PCR reactions to the dye by using
the multichannel pipet and load into the gel.
3.2.14. Bioinformatics Analyses of SAGE Tags
The last step of SAGE is to sequence the colonies and to perform appropriate
bioinformatics analyses on the sequenced products to elucidate SAGE tag
sequences and to calculate relative expression levels of various tags in the sample
(i.e., creation of a SAGE “library”). The frequency of each SAGE tag in the
SAGE library directly correlates with transcript abundance. Sequencing of concatemers
can be done either in-house in a core facility or at any commercial
sequencing laboratory. The bioinformatics associated with analysis of SAGE
tags is outside the scope of this chapter, but the reader is referred to excellent
reviews on this subject (44–46) as well as to relevant websites (SAGENet,
http://www.sagenet.org/; SAGEMap, http://www.ncbi.nlm.nih.gov/projects/
SAGE/; TagMapper, http://tagmapper.ibioinformatics.org/index_html; and
SAGE Genie, http://cgap.nci.nih.gov/SAGE). Of note, many of the databases
house publicly available SAGE libraries of a variety of human tissues, cell
types, and diseased specimens, including pancreatic cancer, and form a readily
available resource for generating differentially expressed transcripts in an entity
of interest.
3.3. Mass Spectrometric Analysis of Proteins
3.3.1. Sample Collection and Preparation of Pancreatic Juice
Human pancreatic juice is normally collected during surgery from the pancreatic
duct of patients undergoing pancreatectomy. A total volume of 20–500 µL
is usually collected and immediately stored at –80°C with or without any protease
inhibitors. The sample should be kept at on ice (4°C) all time when not
stored at –80°C. To remove potential debris the pancreatic juice is centrifuged
at 4°C for 30 min at 16,000g. The protein concentration should be determined