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

188 Cremer and Moos
4. Count cells and divide them into at least 1 × 10 7 cells for RNA extraction and the remaining
cells for DNA extraction. If the cell pellet appears red, resuspend in TE buffer and vortex
for 5 to 10 s to lyse remaining red blood cells, then pellet again quickly and remove
TE buffer.
5. Add 250 µL of Trizol reagent per 1 × 10 7 cells then freeze cells at –20°C for later RNA
extraction. Freeze the remaining cells immediately for later DNA extraction.
6. Thaw pelleted cells in Trizol reagent and extract RNA according to the manufacturers’
instructions.
7. Thaw pelleted cells and isolate DNA, for example using the DNAzol reagent according to
the manufacturers instructions or see Chapter 6 for DNA extraction protocol.
8. Determine concentration of RNA and DNA by optical density (OD) measurement at 260
and 280 nm. Adjust concentration of RNA to 500 ng/µL and of DNA to 100 ng/µL.
9. Confirm integrity of RNA by electrophoresis of 500 ng on a denaturing 1.2% agarose gel
containing 2.2 mM formaldehyde.
10. Confirm integrity of DNA by electrophoresis of 100 ng on a 0.8% agarose gel.
11. Check quality of DNA or RNA by PCR amplifying a gene like β-actin in the case of DNA
or a housekeeping gene like GAPDH in the case of RNA.
3.2. Consensus RT-PCR
1. Perform reverse transcription reaction in a total volume of 20 µL containing 2 µg of RNA
using commercially available kits.
2. Prepare a PCR mixture containing 8 µL of 10× PCR buffer, 1 µL of 20 mM primer
solutions FR1C or FR3A plus LJH-CL, and 2.5 U Amplitaq Gold DNA polymerase.
3. Add the total volume of the RT reaction mixture to the PCR mixture. The final volume
should be 100 µL.
4. Amplify with a program consisting of 7 min of denaturation and enzyme activation at
94°C, followed by 40 cycles of denaturation for 1 min at 94°C and combined annealing
and extension at 63°C (for primer FR3A) or 65°C (for primer FR1C) for 1 min, followed
by a final extension step at 65°C for 5 min.
5. Resolve PCR products either on a 2% (PCR products are approx 350 bp in size if FR1C
was used) or 5% (PCR products are approx 110 bp in size if FR3A was used) ethidium
bromide stained agarose gels. The monoclonal CDR regions lead to a distinct band if the
proportion of myeloma cells is high enough. This band can be distinguished from the
surrounding smear of polyclonal CDR-regions. Figure 2 shows an example of a consensus
PCR (see Note 1).
3.3. Identification of the Myeloma CDR Regions
3.3.1. Cloning of Consensus PCR Products and Direct Lysis
of Transformed Bacteria
1. Excise appropriately sized consensus PCR products from the agarose gel with a scalpel (4).
2. Purify DNA from the agarose block using commercially available kits.
3. Clone-purify PCR products into plasmids and transform competent cells using kits
featuring TA-cloning. If possible, use kanamycin rather than ampicillin for selection, as
satellite colonies occur less frequently. Grow bacteria for 12 to 16 h at 37°C.
4. Pick single colonies and transfer to a replica plate. Spread on an area of approx 1 cm 2 .
Incubate for 12 to 16 h at 37°C. If a distinct band without surrounding smear was visible
after consensus PCR, 10 to 20 colonies should be sufficient. If a band surrounded by a
polyclonal smear was visible, 20 to 40 colonies should be picked.