I have a concern about some patients who co-expressed both p210 and p190 types of BCR-ABL transcripts. In our lab, in first place we perform a real time PCR to detect the expression of Philadelphia chromosome (according to BIOMED 2003), when we obtained a significant amplification of p210 transcript and also a weaker amplification of p190 transcript, we perform a conventional PCR to confirm the co-expression of both transcripts.
My concern is about the analysis of the ethidium bromide-stained agarose gels from this patients. I would like to know which one are the sizes of PCR products that we need to observe to be sure that is positive for p190, in patients who also expressed p210, using the BCR-ABL p190 primer sets A – B from BIOMED-1 study (1999).
The publication indicate this in one of the paragraph: “In all the reactions, after the first round of RT-PCR, the amplified products visualized on ethidium-bromide stained agarose gels showed only single bands of the expected molecular size (Figure 9 and Table 11). However, when testing patients expressing both p190 and p210 types of BCR-ABL transcripts, a very high molecular weight band (approximately 1800 bp) can be observed. This band corresponds to the amplification of the entire cDNA sequences included between the BCR exon 1 and ABL exon 3 in the p210 types of transcripts”.
According to this information, our doubt is, if we need to observed one band of approximately 1800 bp, corresponding to the amplification of p210 transcripts, and additionally to this band, observe a second band of 521bp or 347bp (one round of PCR A-B), corresponding to the amplification of p190 transcripts. Or, the detection of only one band in 1800bp is considered as p190 positive as well??
Thank you for your help
Dear Carolina
You can read this link for useful information about BCR-ABL transcripts identifications by PCR "http://www.msmbb.org.my/apjmbb/html162/162d.pdf"
If you don't have reagents for one-step PCR reaction. You can separately do a mixture for each transcripts with specific primers.
I hope this may be helpfull for you!
Actually, we perform different PCR with different mixes for each transcript, and my question is how can we interpret the result for p190 when we see only one band in 1800bp or two bands in 1800bp and 521 bp. In which of this cases, p190 transcripts are positive?
Thank you for your answer.
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