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. 2015 Mar 12;10(3):e0119256. doi: 10.1371/journal.pone.0119256

Fig 3. Detection of the ETV6-RUNX1 fusion transcript by reverse transcription followed by PCR.

Fig 3

200 ng of RNA purified from umbilical vein cord blood lymphocytes was reverse transcribed using random hexamers as primers. (A) The integrity of the RNA in the numbered cord blood samples was confirmed by amplification of a 384 bp sequence spanning the junction of exons 3 and 4 of the β-2-microglobulin gene. Positive controls included RNA from the BJAB and UoCB4 cell lines (+RT). Negative controls included salmon sperm DNA (SS DNA) and the exclusion of reverse transcriptase (NRT). Lanes containing the 100 bp DNA ladder reference are indicated by M. (B) A 200 bp amplicon obtained from a nested PCR assay indicates the presence of the ETV6-RUNX1 transcript. Representative results show the products of three technical replicates from four numbered cord blood samples, salmon sperm DNA as a negative control, and a 10-2 dilution of the ETV6-RUNX1-positive UoCB4 cells among BJAB cells as a positive control. Samples in which at least two of three technical replicates were detected from cDNA generated independently in two laboratories and in which no product was observed when reverse transcriptase was omitted were considered to contain the ETV6-RUNX1 fusion transcript. (C) The ETV6-RUNX1 amplicon generated from cord blood sample 392 was subjected to sequencing with the primers used to generate the product. The sequencing electropherogram from one of two reactions demonstrates the structure of the expected fusion transcript.