The isolation of high-quality RNA and DNA from various specimens is essential to perform reliable molecular diagnostic assays. In routine diagnostics of hematologic malignancies isolation of high-quality RNA is a prerequisite. We used QIAsymphony technology (QST) using a customized RNA CT 800 V6 protocol for automated semi-high-throughput isolation of RNA from human specimens and compared the results for breakpoint cluster region-c-abl oncogene 1 (BCR-ABL1) quantification by real-time quantitative polymerase chain reaction (RQ-PCR) and detection of JAK2 V617F mutations by reverse-transcriptase PCR (RT-PCR) on QST RNA with RNA isolation performed with our routine manual method using RNA-Bee (RB). QST RNA was isolated with and without the addition of β-mercaptoethanol (BME). Addition of BME to the lysis buffer RLT Plus resulted in consistently lower Ct values in analyses of the reference gene porphobilinogen deaminase (PBGD). Further, the BCR-ABL1 mRNA levels of the QST RNA isolation were highly consistent with RB RNA isolation, only when the lysis buffer RLT Plus in addition contained BME. Moreover, cases of myeloproliferative neoplasms (MPN) with low levels of JAK2 V617F mRNA were even missed in QST when lysis buffer RLT Plus was used, but they were readily detected after addition of BME.