1. Although Amplifluor primers are light-sensitive and long exposure to light should be avoided, the author found little decrease in fluorescence signal after 6 mo of storage at -80°C.

2. The T42 and TSR8 are oligonucleotide targets utilized for the PCR amplification. The working area can easily be contaminated from handling the highly concentrated oligonucleotide preparations. These oligomers should be synthesized

Fig. 2. Standard curve.(Please see Companion CD for a color version of this figure.)

by a DNA synthesizer different from those used for synthesis of other primers. In addition, the oligomer solutions should be prepared using specifically designated pipets in a physically separated work area.

3. All immortalized cultured cells express telomerase. Although the author observed several-fold variation of telomerase activity among different cell lines, any cultured cell lines can be used for this purpose. The volume of CHAPS lysis buffer used is adjusted for the number of cells to be extracted. To determine the volume of CHAPS lysis buffer for each sample, establish cell number by counting or extrapolation from tissue weight.

4. When preparing extracts from tumor samples, add RNase inhibitor to CHAPS lysis buffer prior to the extraction for a final concentration of 100-200 U/mL.

5. The problem most commonly associated with the TRAP assay is the use of sample extracts with high protein concentration. Many samples isolated from human tissues and fluids contain Taq polymerase inhibitor(s). In addition, presence of large quantities of proteins in the reaction mixture can occasionally cause amplification of nonspecific PCR products. Therefore, it is essential to control the amount of input proteins in the assay. Diluting the samples below the recommended maximum protein concentration minimizes most of these problems. See Subheading 3.3. for the effect of sample dilution on accurate analysis of clinical samples.

6. The extracts for the TRAP assay should be quick-frozen on dry ice after each use. Aliquots should not be freeze-thawed more than five times to avoid loss of telomerase activity. In addition, aliquoting reduces the risk of contamination.

7. You may alter the order of step b and steps c, I, and e. add telomerase extracts or controls at the bottom of the PCR reaction tube first, and then carefully add 48 | L of the Master Mix.

0 2 4 6 3 10 12 14 16 IS 20 22 24 26 2S 50 32 34 56 38 40



Fig. 3. Amplification plots. (A) TSR8-FAM window. (B) TSR8-ROX window. (C) Experimental samples (approx 1-5) -ROX window. (Please see Companion CD for a color version of this figure.)


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