John M. S. Bartlett.pdf - Bio-Nica.info

Serial Analysis of Gene Expression 281
refrigerated; a vortex mixer; 50-mL tubes and an appropriate centrifuge; wet and dry ice;
water baths; and incubators, including one for bacterial culture.
2. Working with RNA. The quality and quantity of input RNA is critical to the success or
failure of SAGE. Working with RNA may be difficult, and advice is given in Maniatis
and on Ambion’s web site. Materials should be RNase-free: solutions may be DEPC
treated and RnaseZap ® or other RNase inhibitors may be used on equipment. Check RNA
quality by agarose gel electrophoresis: 0.5 µg of total RNA should yield two clear bands
of ribosomal RNA (4.5 kb and 1.9 kb).
3. Protocols for smaller amounts of starting material (see Subheadings 2.2., 2.4., 2.6.,
and 3.2.). The Johns Hopkins protocol as described here (and used personally) requires
a relatively large amount of input material: ideally, at least 2.5 µg of mRNA, broadly
equivalent to 250 µg of total RNA, 250 mg tissue, or 2.5 × 10 7 cultured cells. This
protocol therefore cannot be used to generate expression profiles where RNA is limited,
for example, tissue biopsies. Various technical modifications now enable SAGE to be
applied to smaller quantities of RNA (3): at least 100-fold (and up to 5000-fold) less may
be needed. SADE (a SAGE Adaptation for Downsized Extracts) (6) uses Dynal’s oligo
dT-coated magnetic beads to capture polyA+ mRNA directly from the total RNA or cell
lysate. (This substitutes for mRNA purification then cDNA synthesis with biotinylated
oligo dT followed by capture onto streptavidin-coated Dynabeads.) All steps from
mRNA isolation to tag release are then performed directly on the beads. This procedure
significantly reduces sample loss and has been adopted in Invitrogen’s I-SAGEkit. Oligo
dT is used to similar effect in microSAGE (7) and miniSAGE (8), in the form of a coating
inside microcentrifuge tubes (Roche’s Streptavidin-Coated Tubes). The references contain
the experimental protocols. Further modifications include additional PCR steps. In SADE
and microSAGE, the ditags generated by the first round of large-scale PCR amplification
are then re-amplified using extra PCR cycles (6,7). In contrast, SAGE-Lite (9) and PCR-
SAGE (10) have adapted Clontech’s SMART system to generate PCR-amplified cDNA,
to increase the amount of input material before proceeding to SAGE proper.
4. Enzymatic kinasing of linker oligonucleotides. Dilute linkers to 350 ng/µL. Set up two
tubes, one for linker pair 1 and the other for pair 2. Mix 9 µL of Linker B (either 1B or 2B)
with 8 µL of LoTE, 2 µL of 10× ligase buffer (works with kinase and contains ATP) and
1 µL of T4 polynucleotide kinase. Incubate at 37°C for 30 min then heat inactivate at 65°C
for 10 min. Add 9 µL of Linker 1A to the 20 µL of kinased Linker 1B, and do likewise for
Linkers 2. To anneal linkers, heat to 95°C for 2 min, then place at 65°C for 10 min, 37°C
for 10 min, and room temp for 20 min. Store at –20°C. The final linker concentration is
200 ng/µL. Test the kinase reaction by self-ligating 200 ng of each linker pair. Run on a 12%
polyacrylamide gel. Kinased linkers should result in linker-linker dimers of 80 to 100 bp,
whereas unkinased linkers should not self-ligate. Only linker pairs resulting in over 70%
self-ligation should be used in further steps.
5. Phenolchloroform extraction. If the sample volume is below 200 µL, increase it to 200 µL
with LoTE in a 1.5-mL microcentrifuge tube. Add an equal volume of P/C. Vortex and centrifuge
at full speed for 5 min at room temp. Transfer the aqueous (top) phase to another tube.
6. Ethanol precipitation. For a 200-µL sample volume in a 1.5-mL tube, add 3 µL glycogen
(as a carrier), 100 µL 10 M ammonium acetate, and 700 µL 100% ethanol. (Scale volumes
up or down as required.) Mix well. Precipitate on dry ice (or at –20°C) for at least 15 min.
Centrifuge at full speed for 15 min, preferably at 4°C. Wash pellet with 70% ethanol and
respin. Resuspend in LoTE.
7. QIAquick columns (see Subheading 2.3.). In this protocol, DNA is prepared by P/C extraction
and ethanol precipitation. At some stages, QIAquick ® columns can be substituted,
saving time and possibly providing purer samples for SAGE (12).